Caracterização estrutural de três lectinas apresentando especificidades por açúcar distintas, isoladas de sementes de Fruta Pão (Artocarpus incisa L.)

Abstract

Three lectins from Artocarpus incisa L. seeds, showing different sugar-binding properties, were efficiently isolated and purified by affinity chromatography. The D-galaetose-binding lectin was isolated on an Adenanthera pavonina L. cross-linked galactomannan while the D-mannose-binding leetin was isolated on agarose-mannose and the chitin binding lectins was isolated on chitin columns. These lectins were named FRUTALlN, FRUTAPIN e FRUTACHIN,respectively. The three leetins showed hemagglutinating activities against animal and human ABO system red blood cells, although with no apparent group specificity. With respect to their structure, the three leetins showed some peculiarities. When the apparent molecular masses (Mr), determined by gel filtration on Superdex 75 HR, in the presence of D-galactose and D-glucose were determined, while frutalin and frutapin showed values of 60 kDa, frutachin showed a value of 12 kDa. On the other hand, by PAGE-SDS, frutalin showed two bands (15 and 12 kDa) while only one band was found both for frutapin (13 kDa) and frutaehin (6.5 kDa). These data suggest that frutalin and frutapin are tetramers and frutachin a dimmer. Frutalin and frutapin showed a very similar amino acid composition, with high contents of glycine and acidic and hydroxylated residues and absence of cysteine while frutachin showed a very high content of this amino acid (more than 14%). The secondary structures of the three leetins were showed to be predominantly B, with no significant a-helix, although frutapin and frutachin did not show CD spectra characteristics of B-structure. The frutalin and frutaehin N-terminal amino acid sequences were determined and submitted to alignment (BLAST data bank); while frutalin showed more than 90% of identity with jacalin, frutachin showed identity with no leetin deseribed. Thus frutachin may constitute a new lectin family. The stability of frutalin quaternary structure, with to pH, was investígated by gel filtration, in the presence and absence of D-glucose and D-galactose. The presenee of these sugars stabilized the strueture of the lectin. The effects of these two sugars, structurally non related, suggest that frutalin must recognize both monosaccharides, and allow us to define a new subdivision for the leetins, the MULTILECTINS, or proteins composed by carbohydrate-binding domains, whieh are identical or highly homologous, but recognizing structurally non related sugars. Thus, lectins showing multivalent specificity should be induded in this class.