PURIFICATION AND CHARACTERIZATION OF ALPHA-L-FUCOSIDASE FROM CHINESE-HAMSTER OVARY CELL-CULTURE SUPERNATANT

In this study, alpha-L-fucosidase from Chinese hamster ovary (CHO) cel l culture supernatant was purified 11 200-fold to apparent homogeneity to assess the rate of fucose hydrolysis from oligosaccharide and glyc oprotein substrates. The fucosidase migrated as a single band of 51 kD a on SDS-PAGE and is a glycoprotein, as determined by retention on con canavalin A-Sepharose, and by lectin blotting with concanavalin A. Hyd rolysis of the artificial substrate 4-methylumbelliferyl-alpha-L-fucos ide (4MU-Fuc) followed simple Michaelis-Menten kinetics, and was compe titively inhibited by free fucose and by two known fucosidase inhibito rs, fucosylamine and deoxyfuconojirimycin. Hydrolysis of fucose from o ligosaccharides including 2'-fucosyllactose, 3-fucosyllactose, Fuc alp ha(1,6)GlcNAc and pooled gp120 oligosaccharides with the Fuc alpha(1,6 )GlcNAc linkage also followed simple Michaelis-Menten kinetics. Howeve r, activity toward 4MU-Fuc was optimal near pH 7, while activities tow ard the oligosaccharide substrates were optimal near pH 5. No fucose w as. released from the recombinant CHO cell-produced glycoproteins gp12 0 or soluble CD4 with the Fuc alpha(1,6)GlcNAc linkage, or from human serum alpha(1)-acid glycoprotein with the Fuca(1,3)GlcNAc linkage. Enz ymatic removal of sialic acid and galactose from gp120 oligosaccharide s did not alter the susceptibility of gp120 to fucosidase attack. Thes e data suggest that released CHO cell fucosidase does not contribute t o the heterogeneity of fucosylation that has been observed in CHO cell culture-produced glycoproteins.