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Background: The ability of lectins to bind carbohydrates makes them powerful tools to explore a myriad of biological structures and processes. The Pterocarpus (family: Leguminosae, subfamily: Papilionoideae, tribe: Dalbergieae) seed lectins are mannose/glucose specific, have the same molecular weight and subunit composition, but show slight differences in their fine specificity and affinity for oligosaccharides. Aims: Sequencing, modeling and heterologous expression of the gene encoding for the seed lectin of three Pterocarpus spp: P. indicus, P. officinalis and P. echinatus. Methods: A strategy of directed PCR amplification from their genomic DNA was followed. Primers used were originally designed for P. angolensis seed lectin, whose sequence was previously determined (NCBI, accession numbers CAD19803-11). The known 3D-structure of the P. angolensis seed lectin (determined by X-ray crystallography) was used to build a model for the three Pterocarpus spp. lectins using the 'Swiss-model' programs (http://swissmodel.expasy.org/). The DNA fragment encoding for the mature protein was cloned into the expression vector pBADMycHis and transformed into E. coli TOP10 cells for expression and purification by affinity chromatography. Results: The sequences yielded a single DNA fragment, without introns, coding for the mature protein and a leader sequence. Alignment of the putative amino acid sequence of the three Pterocarpus spp. and the known sequence of P. angolensis seed lectins showed that, as for P. angolensis, the four amino acid residues that are known to be essential for sugar-binding are also conserved in the three Pterocarpus spp., i.e. a triad of an Asp 86 (in loop A), Gly106 (in the extended omega loop), and Asn 138 together with an aromatic residue Phe 132 (in the metal binding loop). The predicted structures are identical to the P. angolensis lectin. Residue 221, which is an Ala in the three Pterocarpus species but a Glu in P. angolensis, is part of the specificity loop and is located very closely to the primary binding site. Moreover, P. officinalis substitutions Ser104 and Gly141 (respectively a Gly and an Asp in P. angolensis) belong to the omega-loop and to the metal-biding loop respectively. The expressed protein from P. indicus and P. echinatus shows similar but not identical carbohydrate-binding properties when compared to the seed lectin. Conclusion: Pterocarpus lectins share a high percentage of identity at the amino acid level which makes them interesting as natural mutants to study the role of specific amino acid mutations in the affinity of the lectins. |
