Fabry disease is an X-linked lysosomal storage disease that results from a deficiency in the enzyme α-galactosidase (α-GAL). α-GAL hydrolyzes α-galactosides, and patients with Fabry disease suffer from an accumulation of these undegraded substrates. Human α-GAL naturally occurs as a homodimer, as determined through SEC and crystallographic analysis. This means its quaternary structure consists of two identical α-GAL subunits that are associated together into a single unit. Other species, such as rice, produce a monomeric form of α-GAL, consisting of only a single subunit. If α-GAL is functional as both a homodimer and monomer, then how does homodimerization affect the activity of human α-GAL? This can be answered through two model systems. First, a monomeric form of human α-GAL can be produced, testing the activity of human α-GAL in a monomeric state. A variant of α-GAL was engineered (called α-GALF273G/W277G) that appeared promising. Secondly, another system can be produced capable of stabilizing one active site of the dimer and testing the other active site for activity. Another lysosomal enzyme, α-N-acetylgalactosaminidase (α-NAGAL), shares 46% amino acid sequence identity and share 11 of 13 active site residues. Previously, an α-GAL variant (called α-GALE203S/L206A) was produced, that maintained the antigenicity of α-GAL, but had acquired the enzymatic specificity of α-N-acetylgalactosaminidase (α-NAGAL). A heterodimeric form of α-GAL can be produced combining one subunit of α-GAL with the engineered variant. The engineered site can be stabilized, while the wild-type site can be tested for activity. SEC analysis suggests α-GALF273G/W277G is a monomer, and its kinetic properties are reported. Evidence shows monomeric α-GAL could be useful as an improved enzyme replacement therapy. Western blotting and activity assays suggest the presence of the α-GAL/ α-GALE203S/L206A heterodimer.

Fabry disease (FD) is an X-linked recessive lysosomal storage disorder (LSD) known to affect approximately 1 in every 40,000 males, and a smaller number of females. FD results from a deficiency of functional α-galactosidase (α-GAL), which leads to the accumulation of terminally α-galactosylated substrates in the lysosome. The predominant treatment is Enzyme Replacement Therapy (ERT), requiring the regular infusion of recombinant human α-GAL. More than half of individuals receiving ERT experience a range of adverse infusion reactions, and it has been reported that as many as 88% of patients receiving ERT develop neutralizing IgG antibodies against the drug. In aim of designing a non-immunogenic treatment candidate for Fabry disease ERT, we have engineered the active sites of α-GAL and another homologous family 27 exoglycosylase named α-N-acetylgalactosaminidase (α-NAGAL) to have interconverted substrate specificities. 11 of 13 active site residues are conserved between these two enzymes, and we have shown that their substrate specificities can be interconverted by mutating the two non-conserved active-site residues. We report the kinetic properties of these two mutants along with wild type controls, and use western blotting to show that both mutant enzymes retain their respective wild type enzyme antigenicity. Structural data obtained by X-ray crystallography on the α-GAL mutant (called α-GALSA ) reveals the mechanism by which substrate specificity is dictated between these two proteins, and provides explanations for the mutant’s reduced catalytic efficiency.