PURIFICATION TO HOMOGENEITY OF CHARONIA-LAMPAS ALPHA-FUCOSIDASE BY USING SEQUENTIAL LIGAND-AFFINITY CHROMATOGRAPHY

An alpha-fucosidase from the liver of the marine gastropod Charonia lampas was purified to homogeneity using a procedure that included cation-exchange and gel-filtration chromatography, chromatofocusing and a final series of affinity-chromatography steps which involved the following gel-immobilized ligands: N-(5-carboxy-1-pentyl)-1,5-dideoxy-1,5-imino-L-fucitol, N-(5-carboxy-1-pentyl)-2-acetamido-1,5-imino-1,2,5-trideoxy-D-glucitol and thio-beta-D-galactoside. The enzyme was found to be a tetrameric glycoprotein with a native M(r) of 208000, and to exist in a number of isoforms displaying pI values in the range 6.0-6.4. Substrate-specificity studies using a number of fucosylated oligosaccharides of the lacto-N and lacto-N-neo series and a synthetic disaccharide confirmed that the enzyme catalyses the hydrolysis of a broad range of fucosidic linkages, and established the following hierarchy of susceptibility: Fuc-alpha-2Gal-beta-4Galc >>> Fuc-alpha-6GlcNAc > Fuc-alpha-2Gal-beta-4GlcNAc > Gal-beta-3(Fuc-alpha-4)GlcNAc >>> Gal-beta-4(Fuc-alpha-3)GlcNAc. Similar relative rates of hydrolysis were also demonstrated using biantennary oligosaccharide alditols as substrates which contained fucose linked either alpha-3 or alpha-6 to the N-acetylglucosaminitol residue of the chitobiosyl core.