Enzymatic activity of alpha-L-fucosidase and L-fucokinase across vertebrate animal species

Abstract

The oligosaccharide portion of glycoproteins is known to modulate protein structure, function, and turnover. Our laboratory is interested in the metabolism of L-fucose, a normal constituent of eukaryotic glycoproteins. L-fucose is unique in that it is the only levorotatory sugar utilized in mammalian systems. There is considerable interest in understanding the controls which determine the level of L-fucose attached to proteins, in order to generate stable and active glycoforms of protein for the treatment of disease. As part of a program to determine the controls on protein L-fucosylation, we have systematically determined the tissue distribution of the enzymes L-fucokinase and alpha-L-fucosidase in species across the vertebrate animal kingdom. In general, the level of alpha-L-fucosidase is higher than L-fucokinase level. The tissue with highest enzyme activity cannot be generalized, regardless of which enzyme is of interest. Furthermore, there is not a correlation between synthetic and catabolic enzyme activity within a tissue. L-fucokinase can be detected in all tissues examined. Interestingly, we have also detected ss-D-fucosidase activity, present in extraordinary levels in the liver and small intestine of snake. Whether this is due to a specific enzyme or whether it represents a broad specificity of the alpha-L-fucosidase is currently being investigated.

Fig. 1

L-Fucokinase and α-L-Fucosidase Activity in Birds. A. Tissues of pigeon (Columba livia), bobwhite quail (Colinus virginiana) and ringneck pheasant (Phasianus colchicus) were isolated, extracted and centrifuged. The resulting supernates were reacted with L-fucokinase cocktail containing L-[5,6-³H]fucose for 1 h. Samples were then spotted on DE-81 filters and L-[5,6-³H]fucose-1-phosphate determined after binding, washing and scintillation counting. Each data point is the mean activity per gram tissue wet mass, ± sem of three animals assayed in duplicate. B. Identical aliquots of tissue supernates were reacted with 1 mM p-nitrophenyl-α-L-fucopyranoside in BSA/citrate buffer, pH 5.0 for 1 h – overnight. Samples were then terminated and measured by spectrophotometry using an ε= 18.5 × 10³ M. Each data point is the mean activity per gram tissue wet mass ± sem of three animals assayed in duplicate.

Fig. 2

L-Fucokinase and α-L-Fucosidase Activity in Rodents. A. Tissues of jackrabbit (Lepus californicus) and cottontail rabbit (Sylvilagus floridanus) were isolated, extracted and centrifuged. The resulting supernates were reacted with L-fucokinase cocktail containing L-[5,6-³H]fucose for 1 h. Samples were then spotted on DE-81 filters and L-[5,6-³H]fucose-1-phosphate determined after binding, washing and scintillation counting. Each data point is the mean activity per gram tissue wet mass, ± sem of three animals assayed in duplicate. B. Identical aliquots of tissue supernates were reacted with 1 mM p-nitrophenyl-α-L-fucopyranoside in BSA/citrate buffer, pH 5.0 for 1 h – overnight. Samples were then terminated and measured by spectrophotometry using an ε= 18.5 × 10³ M. Each data point is the mean activity per gram tissue wet mass ± sem of three animals assayed in duplicate.

Fig. 3

L-Fucokinase and α-L-Fucosidase Activity in Fish. A. Tissues of largemouth bass (Micropterus salmoides), carp (Cyprinus carpio), and channel catfish (Ictalurus punctatus) were isolated, extracted and centrifuged. The resulting supernates were reacted with L-fucokinase cocktail containing L-[5,6-³H]fucose for 1 h. Samples were then spotted on DE-81 filters and L-[5,6-³H]fucose-1-phosphate determined after binding, washing and scintillation counting. Each data point is the mean activity per gram tissue wet mass, ± sem of three animals assayed in duplicate. B. Identical aliquots of tissue supernates were reacted with 1 mM p-nitrophenyl-α-L-fucopyranoside in BSA/citrate buffer, pH 5.0 for 1 h – overnight. Samples were then terminated and measured by spectrophotometry using an ε= 18.5 × 10³ M. Each data point is the mean activity per gram tissue wet mass ± sem of three animals assayed in duplicate.

Fig. 4

L-Fucokinase and α-L-Fucosidase Activity in Reptiles. A. Tissues of snapping turtle (Chelhydra serpentina), bull snake (Pituophis cantifer sayi), and bullfrog (Rana catesbeiana) were isolated, extracted and centrifuged. The resulting supernates were reacted with L-fucokinase cocktail containing L-[5,6-³H]fucose for 1 h. Samples were then spotted on DE-81 filters and L-[5,6-³H]fucose-1-phosphate determined after binding, washing and scintillation counting. Each data point is the mean activity per gram tissue wet mass, ± sem of three animals assayed in duplicate. B. Identical aliquots of tissue supernates were reacted with 1 mM p-nitrophenyl-α-L-fucopyranoside in BSA/citrate buffer, pH 5.0 for 1 h – overnight. Samples were then terminated and measured by spectrophotometry using an ε= 18.5 × 10³ M. Each data point is the mean activity per gram tissue wet mass, ± sem of three animals assayed in duplicate.

Fig. 5

Correlation between α-L-fucosidase and L-fucokinase activities. The average of each species’ α-L-fucosidase activity are plotted versus the average of each species’ L-fucokinase activity. The error bars are omitted for clarity.