Luiz Augusto Gomes de Souza

Purification, Chemical, and Immunochemical Properties of a New Lectin from Mimosoideae (Parkia Discolor)

ABSTRACT A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P.discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.

CRLI induces vascular smooth muscle relaxation and suggests a dual mechanism of eNOS activation by legume lectins via muscarinic receptors and shear stress

Lectins are proteins able to recognize carbohydrates, without modifying their structure, via the carbohydrate-recognition domain (CRD). Here, the three-dimensional structure of the mannose-binding lectin isolated from Cymbosema roseum (CRLI) was determined with X-man molecule modeled into the carbohydrate recognition domain. CRLI relaxant activity in thoracic rat aorta was also investigated, and based on the results, a molecular docking of CRLI with heparan sulfate was performed to investigate the possible interaction with mechanoreceptors involved in vasorelaxation. CRLI (IC₅₀=12.4 μg mL(-)(1)) elicited vasorelaxant response (96%) in endothelialized rat aorta contracted with phenylephrine. Endothelium-derived relaxant factors, extracellular calcium (Ca(2+)e) and muscarinic receptors were also evaluated as putative participants in the CRLI relaxant effect. CRLI relaxant effect was blocked by L-NAME, a nonselective inhibitor of nitric oxide synthase (NOS), and partially inhibited in a calcium-free solution (0Ca) and by atropine, but it remained unchanged in the presence of indomethacin and TEA. In summary, our data suggest interaction between CRLI and muscarinic receptors located in vascular endothelial cells leading to NOS activation triggered by a mechanism that involves Ca(2+)e along with the ability of CRLI to interact with heparan sulfate, a highly rated mechanoreceptor involved in eNOS activation.

Purificação parcial de inibidores de tripsina de sementes de Caesalpinia ferrea e Swartzia polyphylla e o efeito dos extratos protéicos sobre fungos fitopatogênicos

Legume seeds present high concentration of trypsin inhibitors; these proteins are involved in both cell metabolism and plant defense mechanisms. In order to confirm or not a possible fungicidal action of these proteins from extracts of seeds of legume tree species, the aim this study was to detect trypsin inhibitors in seeds of Caesalpinia ferrea (CfTI) and Swartzia polyphylla (SpTI) and to test their extracts against the pathogenic fungi Colletotrichum guaranicola, Corynespora cassiicola, Fusarium oxysporum and Sclerotium rolfsii, evaluating mycelial growth and sporulation. Thus, samples of the plant material, finely powdered seeds, were extracted in 150 mM NaCl. The protein extracts were partially purified on Sephadex G-100 column, subjected to detection of inhibitors and SDS-PAGE (12.5%), and used in bioassays against the fungi. The electrophoretic profile revealed a single band in CfTI and eight bands in SPTI. The extracts of C. ferrea and S. polyphylla had an effect in reducing sporulation of the tested fungi, but S. rolfsii was inhibited only by C. ferrea. As to mycelial growth, the two extracts showed an effect on F. oxysporum and S. rolfsii, while C. guaranicola was inhibited only by S. polyphylla, and C. cassiicola by C. ferrea. It was concluded that seeds of C. ferrea and S. polyphylla present trypsin inhibitors. In addition, the results suggest that these tropical tree species are promising for the exploration of natural fungicides, since the extracts reduced mycelial growth and sporulation of C. guaranicola, C. cassiicola, F. oxysporum and S. rolfsii.

Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia boliviana Piper seeds.

Plant lectins are the most studied group of carbohydrate-binding proteins. Despite the high similarity between the members of the Diocleinae subtribe (Leguminosae) group, they present differing biological activities. Canavalia boliviana lectin (Cbol) was purified using a Sephadex G-50 column and crystallized in the presence of X-Man by hanging-drop vapour diffusion at 293 K. After optimization, crystals suitable for diffraction were obtained under the condition 0.1 M HEPES pH 7.5 and 3.0 M sodium formate. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 126.70, b = 66.64, c = 64.99 A, alpha = 90.0, beta = 120.8, gamma = 90.0 degrees . Assuming the presence of a dimer in the asymmetric unit, the solvent content was estimated to be about 46%. A complete data set was collected at 1.5 A resolution.

Seeds of Amazonian Fabaceae as a source of new lectins

Seeds from fifty native Amazonian Fabaceae species (representing subfamilies Caesalpinioideae, Mimosoideae and Faboideae) were screened for the presence of new lectins. Their crude protein extracts were assayed for hemagglutinating activity (HA). The protein fractions of Anadenanthera peregrina, Dimorphandra caudata, Ormosia lignivalvis and Swartzia laevicarpa exhibited HA, and this activity was inhibited by galactose or lactose but not by glucose or mannose. The crude extract of S. laevicarpa exhibited HA activity only after ion exchange chromatography, and its lectin was further purified by affinity chromatography on immobilized lactose. Despite the large number of lectins that have been reported in leguminous plants, this is the first description of lectins in the genera Anadenanthera, Dimorphandra and Ormosia. The study of lectins from these genera and from Swartzia will contribute to the understanding of the evolutionary relationships of legume lectins in terms of their protein processing properties and structures.

Prospeção de inibidores de serinoproteinases em folhas de leguminosas arbóreas da floresta Amazônica

The proteinase inhibitors are proteins extensively investigated in tissue storage, but few prospected in other plant tissues. The aim of this study was to detect the presence of serine proteinase inhibitors in leaf extracts from fifteen species of leguminous trees of the Amazon forest. The species studied were Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata and S. polyphylla. Leaves were collected, dried at 30oC for 48 h, crushed and subjected to extraction with NaCl (0.15 M, 10% w/v), resulting in the total extract. Tests were performed to determine the concentration of proteins and to detect of inhibitory activity against bovine trypsin and chymotrypsin. The content of crude and soluble protein in leaf extracts varied from 7.9 to 31.2% and 1.3 to 14.8%, respectively. The inhibitory activity on trypsin and chymotrypsin was observed in all leaf extracts. However, in extracts of E. maximum, L. leucocephala, P. pendula, S. corrugata and S. polyphylla, the inhibition was greater on trypsin, while extract of P. multijuga was more effective against chymotrypsin. We conclude that leaf extracts of leguminous trees have serine proteinase inhibitors and show potential biotecnological applications.

Partial purification of trypsin inhibitors from Parkia seeds (Fabaceae)

Leguminous seeds (Fabaceae) have a high content of inhibitors of which serine protease inhibitors are the most widely studied. However, there are only a few studies related to the investigation of these proteins in tree species belonging to the Amazon flora. The protein content presented in seeds of four Amazonian Leguminosae species, Parkia pendula, P. discolor, P. multijuga and P. Nitida, was extracted by using NaCl 0.15 mol L-1 and then partially fractionated by using affinity chromatography performed on a trypsin-Sepharose 4B. These inhibitors presented different affinities between trypsin and chymotrypsin serine proteases, showing a higher inhibition to trypsin compared to chymotrypsin, except for P. nitida, which showed high inhibition against both enzymes. The SDS-PAGE analysis showed that the species from Parkia genus have a main band corresponding to partially purified trypsin inhibitors. The apparent molecular mass inhibitors (approximately 13-18 kDa) and the high specificity for trypsin suggest the occurrence of Bowman-Birk and Kunitz type inhibitors.

Características químicas de solos de várzeas sob diferentes sistemas de uso da terra, na calha dos rios baixo Solimões e médio Amazonas

The Solimoes/Amazonas River channel high and low land floodplain soils were studied on Manacapuru, Iranduba, Itacoatiara and Silves townships, with purpose the evaluate the chemical characteristics of floodplain soils, as well as, the effect of land use system on soil nutrient status. Nineteen different systems were sampled, eight in the low Solimoes river and eleven in medium Amazonas river. Sampling was performed on 0-10, 10-20 and 20-40 cm deep upper soil layers. Contents of pH, Al, Ca, Mg, K, P, C, N, Zn, Cu, Mn, and Fe, were determined. All the land use shows high available of Ca, Mg, P, Zn, Cu, Mn e Fe. Despite the majority soil samples presented high contents of K, the secondary forest and agroforestry systems presented medium content of K, demonstrated this nutrient can be deficient in some systems in the floodplain soils. The systems of forest and secondary forest presented more acidity and higher available Al, contracting with the others systems land use. C and N levels were lower confirming N to be one of the main limiting factors on the farming yield in the Amazonian floodplains.

A novel vasorelaxant lectin purified from seeds of Clathrotropis nitida: partial characterization and immobilization in chitosan beads.

A novel lectin from seeds of Clathrotropis nitida (CNA) was purified and characterized. CNA is a glycoprotein containing approximately 3.3% carbohydrates in its structure. CNA promoted intense agglutination of rabbit erythrocytes, which was inhibited by galactosides and porcine stomach mucin (PSM). The lectin maintained its hemagglutinating activity after incubation in a wide range of temperatures (30-60 °C) and pH (6.0-7.0), and its binding activity was dependent on divalent cations (Ca(+2) and Mg(+2)). SDS-PAGE showed an electrophoretic profile consisting of a single band of 28 kDa, as confirmed by electrospray ionization mass spectrometry, which indicated an average molecular mass of 27,406 ± 2 Da and the possible presence of isoforms and glycoforms. In addition, CNA exhibited no toxicity to Artemia sp. nauplii and elicited reversible and dose-dependent vasorelaxation in precontracted aortic rings. CNA was successfully immobilized on chitosan beads and was able to capture PSM in solution. This study demonstrated that CNA is a lectin that has potential as a biotechnological tool in glycomics and glycoproteomics applications.

Crecimiento inicial de Palo de Rosa (Aniba rosaeodora Ducke) en distintos ambientes de fertilidad

Studies of nutritional requirements are of great importance for identifying the most important nutrients in physiologic development and seedling growth. A greenhouse pot experiment was conducted at INPA to evaluate the mineral nutritional demands and the effects of macronutrient omission in the plant growth of Aniba rosaeodora Ducke. The following treatments were used: Complete (fertilization with N, P, K, Ca, Mg, S, B and Zn), Standard (nature soil), Complete without N, Complete without P, Complete without K, Complete without Ca, Complete without Mg, and Complete without S. An Ultisoil with low nutrient availability was used as a substratum. The following characteristics were evaluated, relative growth rate (RGR), net assimilation rate (NAR), plant height, diameter, dry matter production of the aerial part (DMPAP) and amount of nutrient in the dry matter of leaves. We concluded from the results that: low availability of N, Ca and Mg constraints the growth of the Aniba rosaeodora plants. Seedlings of A. rosaeodora required little P, K and S. The omission of Ca and N harmed the RGR of the species. The most important elements for DMPAP were Ca and Mg; the Mg acting more in the leaf area; on the other hand, the omission of sulfur favored the macronutrient absorption (N, P, K, Ca, Mg).