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Investigation of the Effect of Dimerization on Human α-Galactosidase Activity

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dc.contributor.advisorScott C Garman
dc.contributor.authorDooley, Scott R
dc.contributor.departmentUniversity of Massachusetts Amherst
dc.contributor.departmentBiochemistry
dc.date2023-09-23T08:33:51.000
dc.date.accessioned2024-04-26T20:37:59Z
dc.date.available2014-06-14T00:00:00Z
dc.date.issued2014-01-01
dc.date.submittedFebruary
dc.description.abstractFabry 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.
dc.description.degreeMaster of Science (M.S.)
dc.identifier.doihttps://doi.org/10.7275/4927160
dc.identifier.urihttps://hdl.handle.net/20.500.14394/44620
dc.relation.urlhttps://scholarworks.umass.edu/cgi/viewcontent.cgi?article=2339&context=theses&unstamped=1
dc.source.statuspublished
dc.subjectα-Galactosidase
dc.subjectactivity
dc.subjectmonomer
dc.subjectheterodimer
dc.subjectdimerization
dc.subjectBiochemistry
dc.subjectMolecular Biology
dc.subjectOther Biochemistry, Biophysics, and Structural Biology
dc.subjectStructural Biology
dc.titleInvestigation of the Effect of Dimerization on Human α-Galactosidase Activity
dc.typecampus
dc.typearticle
dc.typethesis
digcom.contributor.authorisAuthorOfPublication|email:srdooley@student.umass.edu|institution:University of Massachusetts Amherst|Dooley, Scott R
digcom.date.embargo2014-06-14T00:00:00-07:00
digcom.identifiertheses/1177
digcom.identifier.contextkey4927160
digcom.identifier.submissionpaththeses/1177
dspace.entity.typePublication
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