Mutagenesis of Human Alpha-Galactosidase A for the Treatment of Fabry Disease

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme, α-galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40-60 years of age. There is currently only one FDA approved treatment (Fabrazyme) classified as an enzyme replacement therapy. However, approximately 88% of patients experience a severe immune response that, rarely, can be fatal and is a huge cost burden at average $250,000 a year per patient. The structure of α-galactosidase A has been previously determined to be a homodimer with six N-linked glycosylation sites, and the catalytic mechanism determined to be a ping-pong bi-bi with the second substrate being water. The purpose of this research is twofold: first, to generate a more efficient enzyme replacement therapy alternative; and second, to develop an alternative assay to detect the activity of the enzyme that can lead to a better screen for the presence of lysosomal storage disorders in patients. A more efficient therapy was investigated utilizing two different host expression systems, Escherichia coli and Pichia pastoris, as well as site directed mutagenesis of the human enzyme. Detectable expression was not observed in E. coli, so mutagenesis was carried out using P. pastoris. Three locations on the structure of α-galactosidase A protein were targeted for mutation the active site, the dimer interface, and the hydrophobic loop between the active site and the third glycosylation site. v Viable candidates for further study into a therapeutic α-galactosidase A were determined based on catalytic efficiency (kcat/Km), thermostability, and possible glycosylation independence. Three mutations were identified to be of potential therapeutic significance in each one of the targeted areas. A continuous enzyme assay was developed with an artificial fluorescent substrate, 4-methylumbelliferyl-α-D-galactopyranoside, that at acidic pH generates the same kinetic values as the preexisting discontinuous time point assay at basic pH. This development has not only potential laboratory benefits if implemented; it also has a clinical significance in screening for Fabry disease and can potentially be extended to other lysosomal storage disorders