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Lectins are a group of carbohydrate-binding proteins or glycoproteins that are widely distributed in nature. Due to their ability to bind carbohydrates, lectins are thus powerful tools for the isolation and analysis of glycoconjugates. In the current work, a lectin from seeds of Pterocarpus indicus (family: Leguminosae, subfamily: Papilionoideae, tribe: Dalbergieae) was isolated and purified using a combination of ammonium sulfate precipitation and affinity chromatography. The P. indicus seed lectin was grouped under the Man/Glc-specific subgroup of legume lectins, having higher specificity for mannose than for glucose. Results from SDS-PAGE and gel filtration chromatography reveal that P. indicus seed lectin is built up of two monomers of identical molecular mass (28 kDa), just as the seed lectins from other species from the same genus, P. angolensis, P. echinatus and P. officinalis. The gene encoding the P. indicus seed lectin was sequenced, starting from the total genomic DNA and using primers originally designed for P. angolensis seed lectin, whose sequence was previously determined (NCBI protein sequence database, accession numbers CAD19803-11). A single gene, without introns, coding for a signal peptide and for a polypeptide of 252 amino acids, was amplified. Alignment of the putative amino acid sequences of P. indicus and P. angolensis seed lectins reveals 97% identity. Based on the known 3D-structure of the P. angolensis seed lectin (determined by X-ray crystallography) a reliable model could be built for the P .indicus lectin using the Swiss-model programs. The structure obtained is completely similar to the structure of the P. angolensis lectin. In this model the four amino acids that are known to be involved in sugar-binding (Gly106, Asp86, Asn138 and Phe132) are occupying exactly the same position in space. The P. indicus lectin contains a cysteine at position 176, but this residue is not likely to influence the sugar-binding properties. On the other hand, Ala221 in P. indicus (a Phe in P. angolensis), being in the immediate vicinity of the binding site, might affect the sugar-binding. This substitution may be supposed to contribute to the marked differences in strength of adsorption of both lectins to the affinity matrix used for their purification. The DNA fragment encoding for the mature protein was cloned into the expression vector pBADMycHis and transformed into E. coli TOP10 cells. The expressed protein was purified, characterized and compared with the lectin purified from the seeds. The expressed protein has the same molecular weight as that of the seed lectin and shows also similar but not identical carbohydrate-binding properties. The CD spectra of both seed and recombinant lectin from P. indicus were determined, both in the far- and the near-UV region. The spectra of the expressed recombinant lectin do not differ from the spectra of the P. indicus seed lectin. In addition the CD spectra of P. indicus lectin are identical to the spectra of P. angolensis lectin in both regions. |
