SEED LECTINS FROM THE SUB-TRIBE DIOCLEINAE (LEGUMINOSAE- PHASEOLAE): A CHEMOTAXONOMIC TOOL Ana Cristina Oliveira Monteiro-Moreira1, Walbert Edson Muniz-Filho3and Ana Cecília Goes Horta2, Renato Azevedo Moreira* 1 Curso de Ciências Farmacêuticas, CCS, Universidade de Fortaleza 2Laboratório de Lectinas e Glicoconjugados, Departamento de Bioquímica e Biologia Molecular, Centro de Ciências, Universidade Federal do Ceará, Caixa Postal 6020, 60451-970, Fortaleza (CE), Brasil. Fax: (55 85) 288-9789, email [email protected] 3Departamento de Bioquímica, Universidade Federal do Maranhão, São Luis (MA), Brasil *Corresponding author. Abstract Seeds of fourteen species from the genera Canavalia and Dioclea ( Leguminosae - Papilionoideae - Phaseolae - Diocleinae) were investigated with respect to morphologic (size, mass, hylum length) and chemical characteristics. Thus, The total nitrogen content and amino acid composition of the seed flour were determined and these were submitted to extraction at different pH values. The seed extracts were investigated with respect to their content of protein and hemagglutinating activity, fractionated by affinity chromatography and the lectin content evaluated. The isolated lectins were investigated with respect to their amino acid composition, quaternary structure, thermal stability and immunochemical characteristics. The numerical data obtained were submitted to cluster analysis and a phenogram constructed. Key Word Index - Diocleinae, Canavalia, Dioclea, lectin, chemotaxonomy Introduction Ophrestiinae and Phaseolinae (Aymard & Cuello, The Leguminosae is one of the three 1991). The sub-tribe Diocleinae, sometimes classi- largest family of flowering plants, exceeded fied as a separate tribe Diocleae (Hutchinson, only by Compositae and Orchidaceae, and is 1964) is composed of tropical plants and comprises the most economically important family from 13 genera Calapogonium, Camptosema, Cana- the Dicotyledonae, with many species (beans valia, Cleobulia, Collea, Cratylia, Cymbosema, and peas) cultivated as source of food and feed- Dioclea, Galactia, Herpiza, Luzonia Macropsy- ing. They are also botanically full of interest canthus, and Pachyrhizus. The genera Canavalia since their more than 17.000 species constitute and Dioclea are largely spread in the tropics and almost 10% of the flowering plants, being virtu- important sources of animal feeding due to their ally ubiquitous and are present in all the envi- high protein content. The genus Canavalia is di- ronments (except the epiphyte and maritime) in vided in 4 sub-genus: Canavalia, Catadonia, Mau- almost all the continents, except from Artartic naloa and Wenderothia, and the genus Dioclea is (Lackey, 1977; Lackey, 1981). divided in 3 sub-genus: Dioclea, Pachilobium and Three sub-families Caesalpinioideae, Platylobium (Sauer, 1964; Maxwell, 1969; Hey- Mimosoideae and Papilionoideae, are recog- wood, 1971; Aymard & Cuello, 1991). nised (although sometimes as separate families, Numerical taxonomy is understood as the Caesalpiniaceae, Mimosaceae and Papiliona- grouping, by numerical methods, of taxonomic ceae). From the family Leguminosae, the Papil- units on the basis of their character states (Sneath ionoideae is the largest (with two thirds of the & Sokal, 1973). The term includes the drawing of genera and species). is divided in 30 tribes and phyllogenetic inferences from the data by statisti- can be found mainly in the tropics and temper- cal methods. These methods require the use of ate regions. The tribe Phaseolae is sub-divided numerical characters or the conversion of informa- in 8 sub-tribes, Cajaninae, Clitoriinae, Dio- tion about taxonomic entities into numerical quan- cleinae, Erythrininae, Glycininae, Kenediinae, tities. VI Reunião Regional da SBBq – Nordeste. 1 Biochemical data have been used for gladiata, C. maritima, C. plagiosperma) and Dio- numerical taxonomy mainly when they deal clea (D. altissima, D. guianensis, D. rostrata, D. with physiologically key substances. Plant seed violacea, D. virgata), sub-family Diocleinae, were proteins play such role, as they are responsible collected in the State of Ceará and one of them (D. for the plant metabolism, defence and nutrient lehmanii) was kindly supplied by Dr. Gerardo reserve. Lectins (carbohydrate binding proteins, Perez (Universidad Nacional de Bogota, Colmbia). capable of cell recognition and showing high Voucher specimens of the studied samples are homology) are believed to play some of these deposited in the Herbário Prisco Beserra of the roles and varied very little during evolution. Universidade Federal do Ceará. Thus the presence, in related taxa, of seed lec- Erythrocytes. Rabbit blood was obtained by punc- tins makes them a suitable molecular tool for ture of the marginal earl's vein of healthy animals. chemotaxonomic and phyllogenetic investiga- Freund’s complete adjuvant was a product of tions (Toms & Western, 1971; Moreira et al, B.D.H. Acrylamide and methylene bisacrylamide 1991; Lis & Sharon, 1998; Peumans & Van were product of Sigma Chemical Co., Sephadex Damme, 1998). G-50, Superose 12 HR and MW markers were The lectins present in seeds of the tribe from Pharmacia, Uppsala, Sweden. Diocleinae are glucose(mannose)-specific and, The morphologic characteristics were determined thus, can be isolated by affinity chromatography with 100 seeds. The hylum (%) was determined as a on Sephadex G-50 column (Moreira et al, 1983; relation between the hylum length and the total seed Moreira & Cavada, 1984; Oliveira et al, 1991; circumference. Vasconcelos et al, 1991; Moreira et al, 1996; Total nitrogen was determined by the method of Moreira et al, 1997; Monteiro et al, 1998). The Baethgen & Allen (1969), and protein concentra- lectins are classified in the basis of their carbo- tion was obtained by the method of Bradford hydrate specificity (Goldstein & Hayes, 1978). (1976) using bovine serum albumin as standard. Although minor differences have been found, Readings at 280 nm were also employed to deter- even within a particular group, these are less mine protein content of the column eluates. noticeable when the lectins are extracted from Hemagglutinating activity was determined by the botanically related plants (Moreira & Oliveira, method described by Moreira & Perrone (1977), in 1983 a; Moreira & Oliveira, 1983 b), reflecting small glass tubes where to a series of 1:2 dilutions the evolutionary relationships. Thus, very close of the crude extract or purified lectins in 0.15 M similarities have been found in the amino acid NaCl containing 5 mM CaCl2 and 5 mM MnCl2 sequences of the seed lectins from Dioclea (250 µl) were added the same volume of a 2% grandiflora, Dioclea lehmanii, Canavalia ensi- suspension of erythrocytes. The degree of agglutina- formis, Canavalia gladiata and Canavalia mari- tion was monitored visually after the tubes have been tima, all belonging to the sub-tribe Diocleinae left at 37 oC for 30 min and subsequently left at room (Perez et al, 1991). temperature for further 30 min. The hemagglutina- In this paper, a comparative study was tion unit (HU) was defined as the reciprocal of the done using the whole proteins and the lectins in highest dilution still giving a visible agglutination. particular, from the seeds of eight species be- The hemagglutination titre was defined as log2 HU longing to three sub-genera of the genus Cana- and the minimum dose as the minimum amount of valia (sub-genus Wenderothia: Canavalia bi- protein still promoting visible agglutination. carinata; sub-genus Catadonia: Canavalia Protein extraction. The dehulled seeds were ground bonariensis and sub-genus Canavalia: Cana- in a coffee grinder (60 mesh) and the protein extracts valia brasiliensis, C. dictyota, C. ensiformis, C. were prepared by treating the seed meal (1 g) with 20 gladiata, C, maritima and C. plagiosperma) and ml of 0.1 M Glycine-HCl pH 2.6; 0.1 M Na-Acetate six species belonging to the three sub-genera of pH 4.0; 0.2 M Na-Acetate pH 4.0; 0.1 M; Tris-HCl the genus Dioclea. (sub-genus Dioclea: Dioclea pH 6.0; 0.1 M Na-Borate pH 8.0 and 0.1 M Na- guianensis, D. lehmanii, D. virgata; sub-genus Borate pH 10.0 buffers, all containing 0.1 M NaCl. Pachylobium: D. altissima, D. violacea; and The suspensions were left at room temperature for 3 sub-genus Platylobium: Dioclea rostrata). h and spun at 10,000 x g for 20 min. at 7 oC. The clear supernatants were used for the various analyses. Material and Methods Affinity chromatography. This was done as described previously (Moreira et al. 1983). The The species studied, belonging to the genera extracts obtained with 0.1 M borate pH 8,0 buffer Canavalia (C. bicarinata, C. bonariensis, C. containing 0.15 M NaCl were applied to a Sephadex brasiliensis, C. dictyota, C. ensiformis, C. G-50 column (40 cm x 1.6 cm) equilibrated with the VI Reunião Regional da SBBq – Nordeste. 2 same buffer, containing 5 mM CaCl2 and 5 mM determined using the expression of Arrhenius MnCl2. After removing unbound material, the (Dawes, 1972). lectin was desorbed from the column with 0.1 M Amino acid analyses were performed after hy- glucose added to the equilibrium solution. The drolysis of the seed material (flour and lectins) eluates were collected in 3.0 ml fractions and samples in sealed glass tubes under N2, for 24 hr, analysed for hemagglutinating activity and protein with 6 N HCl at 110 oC. After hydrolysis HCl was content. removed by evaporation and the residue was ana- Gel filtration. The purified lectins were submitted lysed in a Pharmacia-LKB Biochrom 20 system. to gel filtration on a Superose 12 HR column, in For Cluster analysis, a basic matrix was con- the absence and presence of 0.1 M glucose, in 0.1 structed with the numerical data using the proce- M Glycine-HCl pH 2.6, 0.1 M Na-Acetate pH dures of Oliveira (1991). The degrees of similarity 4.0, 0.1 M Na-Acetate pH 6.0, 0.1 M Tris-HCl pH found were utilised for the construction of the 8.0 buffers all containing 0.15 M NaCl, 5 mM tentative phenogram. In the case of SDS/PAGE, CaCl2 and 5 mM MnCl2.