Establishment of a heterologous system for the expression of Canavalia brasiliensis lectin: a model for the study of protein splicing

During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed. To investigate whether these events can occur in other eukaryotic organisms, an expression system based on Pichia pastoris cells was established. A DNA fragment encoding prepro-ConBr was cloned into the vector pPICZB, and the recombinant plasmid was transformed in P. pastoris strain GS115. Ten clones were screened for effective recombinant protein production. Based on Western blot analysis of the two clones with the highest level of protein expression: 1) diffuse high-molecular mass immunoreactive bands were produced as early as 24 h after induction; 2) a single-, high-molecular mass protein was secreted into the medium, and 3) a significant fraction of the recombinant polypeptides that cross-reacted with anti-ConBr antibodies comprised a band of approximately 34.5 kDa. Diffuse protein bands with high molecular masses are attributed to hyperglycosylation at the single potential N-glycosylation site located in the linker peptide of prepro-ConBr. In contrast, native ConBr is made up of three polypeptides, the intact alpha chain (aa 1-237) and the fragments beta (aa 1-118) and gamma (aa 119-237), which have apparent molecular masses of 30, 16 and 12 kDa, respectively. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing necessary for the biosynthesis of ConBr.

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins.

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConA looked very similar. However, docking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, revealed conformational changes in side chains of the amino acid residues involved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConA requires conformational chances of its monosaccharide-binding site.

Plant lectins, chemical and biological aspects.

Lectins, carbohydrate-binding proteins of non-immune origin, that agglutinate cells or precipitate polysaccharides and glycoconjugates, are well distributed in nature, mainly in the Plant Kingdom. The great majority of the plant lectins are present in seed cotyledons where they are found in the cytoplasm or in the protein bodies, although they have also been found in roots, stems and leaves. Due to their peculiar properties, the lectins are used as a tool both for analytical and preparative purposes in biochemistry, cellular biology, immunology and related areas. In agriculture and medicine the use of lectins greatly improved in the last few years. The lectins, with few exceptions, are glycoproteins, need divalent cations to display full activity and are, in general, oligomers with variable molecular weight. Although the studies on lectins have completed a century, their role in nature is yet unknown. Several hypotheses on their physiological functions have been suggested. Thus, lectins could play important roles in defense against pathogens, plant-microorganism symbiosis, cell organization, embryo morphogenesis, phagocytosis, cell wall elongation, pollen recognition and as reserve proteins. A brief review on the general properties and roles of the lectins is given.

Purification and Partial Characterization of a Lectin from Phaseolus vulgaris.

A method is presented for the isolation of a lectin from a Brazilian cultivar of the common bean (Phaseolus vulgaris L.), through extraction in acidic (pH 4.2) medium, fractionation with ammonium sulfate, and chromatography on DEAE-cellulose. The lectin was shown to be homogeneous by gel electrophoresis and isoelectric focusing.The molecular weight, determined by osmometry, is 100,250 daltons; the isoelectric point, determined by isoelectric focusing, is pH 5.1; and the extinction coefficient at 280 nm and pH 7 is E(1%) (1 cm) = 7.85.The lectin was shown to be a glycoprotein with 9.11% of neutral sugars and 1.44% of amino sugars. Its amino acid composition was characterized by the absence of methionine and a very low content of half-cystine, with a predominance of acidic and hydroxylated amino acids.The lectin presented no specificity when tested with red blood cells of all the groups of the ABO system.