Purification And Immunomudulatory Activity Assay Of Lectins From Agrocybe Cylindracea, Xylaria Hypoxylon And Xerocomus Spadiceus

A heterodimeric lectin, ACL, with a molecular weight of 31.5 kDa was isolated from the fresh fruiting bodies of the mushroom Agrocybe cylindracea. The molecular weights of its subunits were 16.1 kDa and 15.3 kDa respectively. The lectin was obtained after (NHO2SO4 precipitation, DEAE-cellulose anion exchange chromatography and SP-sepharose cation exchange chromatography. The lectin was adsorbed on DEAE-cellulose in 10 mmol/L Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 150 mmol NaCl. It was adsorbed on SP-Sepharose in 10 mmol/L NH4OAc (pH 4.5) and eluted by approximately 0.19 mol/L NaCl in the same buffer. At the concentration of 0.05mol /L NaOH or 0.025mol /L HC1, the agglutinating activity of ACL was decreased; at the concentration of 0.1 mol /L NaOH or HC1, its agglutinating activity was completely destroyed. The lectin was sensitive to inulin and lactose and stable between 10℃ to 50℃. Al3+ and Fe3+ promoted ACL's agglutinating activity. With hanging drop method, the crystal of ACL was acquired. The crystal reached 2.36 A tested with X-ray diffraction. The lectin exhibited potent mitogenic activity toward mouse splenocytes.Xylaria hypoxylon lectin (XHL), with a molecular weight of 29kDa, was isolated from the fruiting bodies of Xylaria hypoxylon. The lectin is composed of its two same subunits with the molecular weight of 14.4 kDa. With (NHO2SO4 precipitation, DEAE-cellulose anion exchange chromatography, CM-celiulose cation exchange chromatography and Sephadex G-75 gel filtration, the lectin was obtained. XHL was absorbed on DEAE-cellulose in 10 mmol/L PB buffer (pH 7.5) and was eluted by the same buffer containing 150 mmol NaCl. It was unadsorbed on CM-cellulose in 10mmol/L NaAc-HAc (pH 5.4). At the concentration of 0.05mol /L NaOH or 0.025mol /L HC1, the agglutinating activity of XHL was decreased; at the concentration of O.lmol /L NaOH or HC1, its agglutinating activity was completely destroyed. XHL's activity was inhibited by inulin and xylose inhibit Its agglutinating activity was stable between 10℃ to 40℃. The cations of Mg2+. Zn2+and Mn2+ promote its agglutinating activity. The inhibition percentage to S-180 and H-22 entity tumour with XHL is 49.84% and 74.13% respectively. The life span of mouse that were inoculated tumour cells was prolonged, and the body weight increase was slowed down by injection of XHL. Meanwhile, the secretion of IL-2, TNF-a and IFN-r, that were relevant to antitumour, were enhanced by injection of XHL to mouse.The Xerocomus spadiceus lectin, XSL, was isolated from extracts of fruiting bodies of the mushroom Xerocomus spadiceus using a procedure that involved (NH4)2SO4 precipitation, anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin was unadsorbed on DEAE-cellulose and Affi-gel blue gel, adsorbed on CM-Sepharose. The hemagglutinating activity was dwindled to an undetectable level at 80℃. The activity was doubled in the presence of 5mmol/L ZnCl2 or lOmmol/L FeCl3, and quadrupled in the presence of 10mmol/L A1C13. The activity was reduced in the presence of 0.03 mol/L HC1 or 0.05mol/L NaOH. Inulin was able to inhibit the agglutinating activity of the lectin. The lectin was capable ofeliciting an approximately four-fold stimulation of mitogenic response in murine splenocytes.