Structural studies of Pleurotus ostreatus Lectin (POL), a fungal protein of medical interest

Lectins are proteins widely diffused in nature that interact non-covalently with carbohydrates. Of all the mushroom proteins, lectins are probably the most extensively investigated because it has been observed that they can exhibit antitumour activity on human cancer cells. A 373 amino acid lectin was isolated from the fruiting bodies of the edible oyster mushroom Pleurotus ostreatus was isolated since it appears to be able to inhibit the growth of human neoplastic cells. The lectin, named POL, Pleurotus ostreatus lectin, and it was purified using two chromatographic steps: a hog gastric mucin or melibiose column followed by a carboxymethyl-cellulose column or a Sephacryl S-100 gel filtration column. Two alternative ways of elution from the affinity column (with lactose 0.2M and with EDTA 5 mM) gave approximately the same yield (2-5 mg) of protein starting with 500 g of mushrooms. X-ray diffraction data of crystals were collected at various beamlines of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The structure was initially solved by Multiple Isomorphous Replacement (MIR) and it was then refined to a final highest resolution of 2.0 Å. The asymmetric unit contains one monomer with two domains and a total of 22 β-strands: 10 forming the domain closest to the N terminus and 12 nearest the C terminus. The two domains pack face to face and the β-sheet strands are radially arranged around a central tunnel packing face-to-face. The lectin is characterized by a weak β-glucosidase activity (Vmax=87.21±1.5 nmol sec-1 mg-1, kcat=0.044 s-1 and Km=240±0.26 μM) and by the presence of two Calcium atoms coordinated to two sites. POL was tested on human pancreatic cancer cells (MiaPaCa-2) and on HepG2 cells, a perpetual cell line consisting of human liver carcinoma cells, and its therapeutic effect was evident. Recombinant POL, whose expression was optimized in E.coli, crystallized in several conditions but the crystals did not diffract although the enzymatic and hemagglutinating activities were preserved. Encapsulation and/or binding to poly(lactic-co-glycolic acid) nanoparticles of POL might be exploited both to direct PLGA towards tumours and also to prepare POL-filled nanoparticles for therapeutic purposes.