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.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:theses-2339 |
| Date | 01 January 2014 |
| Creators | Dooley, Scott R |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Masters Theses 1911 - February 2014 |
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