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) cell culture supernatant was purified 11 200-fold to apparent homogeneity to assess the rate of fucose hydrolysis from oligosaccharide and glycoprotein substrates. The fucosidase migrated as a single band of 51 kDa on SDS-PAGE and is a glycoprotein, as determined by retention on concanavalin A-Sepharose, and by lectin blotting with concanavalin A. Hydrolysis of the artificial substrate 4-methyl-umbelliferyl-alpha-L-fucoside (4MU-Fuc) followed simple Michaelis-Menten kinetics, and was competitively inhibited by free fucose and by two known fucosidase inhibitors, fucosylamine and deoxyfuconojirimycin. Hydrolysis of fucose from oligosaccharides including 2'-fucosyllactose, 3-fucosyllactose, Fuc alpha(1,6)GlcNAc and pooled gp120 oligosaccharides with the Fuc alpha(1,6)GlcNAc linkage also followed simple Michaelis-Menten kinetics. However, activity toward 4MU-Fuc was optimal near pH 7, while activities toward the oligosaccharide substrates were optimal near pH 5. No fucose was released from the recombinant CHO cell-produced glycoproteins gp120 or soluble CD4 with the Fuc alpha(1,6)GlcNAc linkage, or from human serum alpha 1-acid glycoprotein with the Fuc alpha(1,3)GlcNAc linkage. Enzymatic removal of sialic acid and galactose from gp120 oligosaccharides did not alter the susceptibility of gp120 to fucosidase attack. These data suggest that released CHO cell fucosidase does not contribute to the heterogeneity of fucosylation that has been observed in CHO cell culture-produced glycoproteins.
Gramer
M J
MJ
Department of Chemical Engineering, Stanford University, CA 94305-5025.
Schaffer
D V
DV
Sliwkowski
M B
MB
Goochee
C F
CF
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D003208:Concanavalin A / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media; D007700:Kinetics; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D005644:alpha-L-Fucosidase / Q000302:isolation & purification* / Q000378:metabolism
D003470:Culture Media; D003208:Concanavalin A; D005644:alpha-L-Fucosidase