Biochemical and immunological characterization of a glycosylated alpha-fucosidase from the invertebrate Unio: interaction of the enzyme with its in vivo binding partners.
Mammalian alpha-fucosidase (EC 3.2.1.51) is a lysosomal enzyme that catalyzes the removal of fucose residues from glycosphingolipids and its absence in humans results in a rare metabolic disorder called fucosidosis. Among the invertebrates in the molluscs (Unio) two forms of the enzyme have been reported, a 68 kDa non-glycosylated form and a 56 kDa glycosylated form. The glycosylated form has been purified from the seminal fluid of Unio [Biochem. Biophys. Res. Commun. 234 (1997) 54]. In the present study, the 56 kDa glycosylated form has been purified to homogeneity from the whole body tissue of Unio using a series of chromatographic steps. The purified enzyme migrated as a single protein species in 10% SDS-PAGE. Antibodies to the purified enzyme were raised in a rabbit in order to study its biochemical and immunological properties. The purified enzyme is a glycoprotein that exhibits strong binding to Con A-Sepharose gel and can be deglycosylated by PNGase F enzyme suggesting it to be N-glycosylated. The enzyme has been shown to specifically interact with the mannose 6-phosphate receptor protein (MPR 300) purified from goat and Unio. This specific interaction is discussed in view of its possible in vivo binding partners.
Nadimpalli
Siva Kumar
SK
Laboratory of Protein Biochemistry and Molecular Biology, Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India. drnsk7@yahoo.co.in
Padmanabhan
Nirmala
N
Koduru
Suresh
S
D000818:Animals; D002845:Chromatography / Q000379:methods; D003208:Concanavalin A / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000737:chemistry; D008974:Mollusca; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry; D005644:alpha-L-Fucosidase / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification
D006023:Glycoproteins; D008024:Ligands; D003208:Concanavalin A; D012685:Sepharose; D005644:alpha-L-Fucosidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase