Recombinant Canine α-l-Fucosidase: Expression, Purification, and Characterization

Abstract Canine fucosidosis has proven to be an excellent large animal model both for the equivalent human disorder and, in more general terms, for the central nervous system pathology found in many of the lysosomal storage disorders. Most importantly studies in this animal model were among the first to convincingly show that bone marrow transplantation could successfully modify the course of clinical central nervous system disease and to define some of the important parameters for successful treatment. In order to evaluate other, more generally applicable routes to treatment of central nervous system disease in the lysosomal storage disorders we have expressed recombinant canine α- l -fucosidase (rcFUC) in Chinese hamster ovary and Madin–Darby canine kidney cells to levels of between 2 and 13 mg/liter of culture medium and purified the enzyme to apparent homogeneity by affinity chromatography on fucosylamine-linked agarose. rcFUC is composed of subunits of M r 50 kDa and the native enzyme is a homotrimer of M r 156 kDa. Kinetic properties of rcFUC were similar to those of FUC isolated from both human and dog liver. rcFUC was shown to be effective in correcting the storage phenotype of human fucosidosis cells after endocytosis via the mannose-6-phosphate-receptor-mediated pathway. It was also shown to degrade fucosylated storage products isolated from affected dog brain. The availability of large amounts of rcFUC will allow us to explore ways of extending the proven efficacy of enzyme replacement therapy to the treatment of central nervous system pathology using the fucosidosis dog as a model system.