Isolation and characterization of a lectin with antifungal activity from Egyptian Pisum sativum seeds

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Abstract

A plant lectin isolated in its pure state from the Egyptian seeds of Pisum sativum (PSL) produced two bands in SDS–PAGE (5.53 and 19.3 kDa; i.e. α and β chain) but one peak by gel filtration chromatography on Sephadex G-100, corresponding to 50 kDa, i.e., a dimeric structure of two monomers, each consisting of one α and one β subunit. PSL is a glycoprotein bound with glucose (2 mol/mol of protein) and stabilized by 2 atoms of each of Ca2+ and Mn2+ per molecule of protein. It highly agglutinated human, rabbit and rat erythrocytes but weakly agglutinated chicken erythrocytes, while no agglutination occurred with sheep erythrocytes. Hemagglutination was markedly affected by acidic pH, but was heat stable below 60 °C for 30 min. Among the various tested sugars, PSL agglutination was most inhibited by mannose. PSL is rich in hydroxyl amino acids while totally lacking sulfur amino acids. PSL inhibited the growth of Aspergillus flavus, Trichoderma viride and Fusarium oxysporum.

Introduction

Lectins are naturally occurring glycoproteins that bind carbohydrate residues selectively and non-covalently (Van Damme, Van Damme, Prumans, Barre, & Rouge, 1998). Lectins can be found in all kingdoms of life ranging from viruses through bacteria and plants to animals (Loris, 2002). Simple lectins consist of a small number of subunits, not necessarily identical, of molecular weight usually below 40 kDa. This class comprises practically all known plant lectins in addition to the galectins, a family of galactose specific animal lectins (Lis & Sharon, 1998). Legume lectins represent the largest and most thoroughly studied family of the simple lectins. The members of this protein family consist of two or four subunits (or protomers), either identical or slightly different, each with a single, small carbohydrate-combining site with the same specificity. Most legume lectins contain one ion each of tightly attached calcium and manganese per subunit, which are required for carbohydrate binding (Sharon & Lis, 2002). Mannose specific lectins are widely distributed in higher plants and are believed to play a role in the recognition of high mannose glycans of foreign microorganisms or plant predators (Barre, Bourne, Van Damme, Peamans, & Rouge, 2001). Mannose (or glucose) binding lectins form seeds of the family leguminosae including pea (Pisum sativum), lentil (Lens culinaris), fava bean (Vicia faba), common vetch (Vicia cracca) and the forage legume sainfoin (Onobrychis viciiffolia) have been isolated and well-characterized (Goldstein & Hayes, 1978). Pea lectin of a molecular weight 49 kDa was reported to exist as a dimer, where each monomer built up from two β sheets of seven (curved front face) and six (flat back face) anti parallel strands, respectively, interconnected by turns and loops. A third smaller β-sheet made of five short strands, also referred to as the S-sheet, helps to keep together the front and back sheets. The structures resemble flattened bell-shaped domes containing a shallow pocket at their apex, which forms the carbohydrate-binding site. The bottom of the pocket contains binding sites for bivalent metal ions (Barre et al., 2001, Rudiger and Gabius, 2001). In this report, we describe the purification and characterization of a lectin present in Egyptian P. sativum seeds cultivated under warm climatic conditions and report on its antifungal activity. The lectin carbohydrate binding specificity, molecular structure, and biochemical properties were investigated and compared to other reports on plant lectins.

Section snippets

Lectin source

Garden pea (Pisum sativum, Lincoln variety) seeds were obtained from Legume Research Institute, Agricultural Research Centre, Ministry of Agricultural, Giza, Egypt.

Blood samples

Human blood cells (A, AB, B and O) of healthy doners obtained by intravenous withdrawal using heparinized syringes were kindly offered by the blood bank of General Zagazig Hospital. Rabbit, rat, and sheep blood cells were obtained from the animal house of the Faculty of Veterinary Medicine, Zagazig University.

Fungi

Fusarium oxysporum,

Results

After saline extraction, acetic acid precipitation and dialysis, the crude protein preparation from P. sativum seeds was chromatographed on Sephadex G-100 to yield two adsorbed fractions (Fig. 1), designated as A (β-subunit) and B (α-subunit), respectively, in accordance with Driessche, Foriers, Stroberg, and Kanarek (1976). The purification procedure of lectin from seed extract is summarized in Table 1. The lectin from P. sativum seeds (PSL) showed variable hemagglutinating activities when

Discussion

The aim of the present study was to isolate a lectin from Egyptian P. sativum seeds and investigate its antifungal activity. The lectin could be successfully purified in a single step by affinity chromatography on Sephadex G-100. Loading of prepurified extract on an affinity column followed by washing out the unbound proteins then eluting the bound lectin with 250 mM glucose (Fig. 1) led to increments in the specific activity up to 320 titer/mg corresponding to 80% yield. The final obtained

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