Canine alpha-L-fucosidase in relation to the enzymic defect and storage products in canine fucosidosis.
Canine liver alpha-L-fucosidase was purified to apparent homogeneity by affinity chromatography on agarose-epsilon-aminohexanoyl-fucopyranosylamine. It is composed of multiple forms of a common active subunit of 45-50 kDa, which can aggregate in different combinations to form polymers, predominantly dimers. Antiserum was raised against the purified enzyme. There is negligible residual alpha-L-fucosidase in the tissues of English springer spaniels with the lysosomal storage disease fucosidosis. Although no alpha-L-fucosidase protein was detected by Western blotting or by the purification procedure in the affected tissues, some enzymically inactive cross-reacting material was detected in both normal and affected tissues. This suggests that another protein without alpha-L-fucosidase activity was co-purified with the enzyme. Dog liver alpha-L-fucosidase was precipitated by goat anti-(human liver alpha-L-fucosidase) IgG, indicating homology between the enzymes in the two species. Two purified storage products isolated from the brain of a dog with fucosidosis were used as natural substrates for various preparations of canine liver alpha-L-fucosidase. Analysis of the digestion mixtures by t.l.c. and fast-atom-bombardment mass spectrometry suggests that canine alpha-L-fucosidase acts preferentially on the alpha-(1-3)-linked fucose at the non-reducing end and that removal of alpha-(1-6)-linked asparagine-linked N-acetylglucosamine is rate-limiting in the lysosomal catabolism of fucosylated N-linked glycans.
Barker
C
C
Department of Biochemistry, King's College London, U.K.
Dell
A
A
Rogers
M
M
Alhadeff
J A
JA
Winchester
B
B
D000818:Animals; D015153:Blotting, Western; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005645:Fucosidosis / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D014018:Tissue Distribution; D005644:alpha-L-Fucosidase / Q000276:immunology / Q000378:metabolism*
D007527:Isoenzymes; D005644:alpha-L-Fucosidase