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

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Mutagenesis of Human Alpha-Galactosidase a for the Treatment of Fabry Disease City University of New York (CUNY) CUNY Academic Works All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects 9-2017 Mutagenesis of Human Alpha-Galactosidase A for the Treatment of Fabry Disease Erin Stokes The Graduate Center, City University of New York How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/gc_etds/2338 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] CITY COLLEGE, CITY UNIVERSITY OF NEW YORK MUTAGENESIS OF HUMAN ALPHA-GALACTOSIDASE A FOR THE TREATMENT OF FABRY DISEASE By Erin Stokes A dissertation submitted to the Graduate Faculty in Biochemistry in partial fulfillment of the requirement for the degree of Doctor of Philosophy, The City University of New York 2017 ©2017 Erin Stokes All rights reserved ii Mutagenesis of Human α-Galactosidase A for the Treatment of Fabry Disease By Erin Stokes This manuscript has been read and accepted for the Graduate Faculty Biochemistry in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy. ______________________ David H. Calhoun Date Chair of Examining Committee ______________________ Richard Magliozzo Date Executive Officer Supervisory Committee: Haiping Cheng (Lehman College, CUNY) M. Lane Gilchrist (City College of New York, CUNY) Emanuel Goldman (New Jersey Medical School, Rutgers) Kevin Ryan (City College of New York, CUNY) THE CITY UNIVERSITY OF NEW YORK iii Abstract Mutagenesis of Human α-Galactosidase A for the Treatment of Fabry Disease By Erin Stokes Advisor: Dr. David Calhoun 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. iv 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. v Acknowledgements I would like to foremost acknowledge and thank my mentor, Dr. David Calhoun for his advisement and funding of the research. I would also like to thank my committee Drs. Haiping Cheng, M. Lane Gilchrist, Emanuel Goldman, and Kevin Ryan for their feedback and advice on the project. Mariam Meghdari and Nicholas Gao were invaluable in teaching, troubleshooting, and emotional support. Last, I would like to thank several of the undergraduates who volunteered working in the lab and were helpful in the generation of data: Humerya Hekimoglu, Olga Kapustina, David Geiger, Daniel Villarroel, Shazina Shafique, and Thomas Smith. vi Table of Contents Abstract .................................................................................................................................................... iv Acknowledgements .................................................................................................................................. vi Table of Figures ....................................................................................................................................... ix Table of Tables ........................................................................................................................................ xi Table of Abbreviations ............................................................................................................................ xii Chapter 1 Introduction .................................................................................................................................. 1 Fabry Disease Background and Prevalence: ............................................................................................. 1 Structure and Catalytic Mechanism of α-Gal:........................................................................................... 3 Current Treatments and Challenges: ......................................................................................................... 5 Glycosylation ............................................................................................................................................ 8 Homology ............................................................................................................................................... 10 Expression in Pichia pastoris ................................................................................................................. 12 Site Directed Mutagenesis ...................................................................................................................... 12 Important Therapeutic Properties............................................................................................................ 12 Chapter 2 Expression of Human α-Gal in E. coli ....................................................................................... 15 Overview ................................................................................................................................................. 15 E. coli Expression Strains ................................................................................................................. 15 E. coli Expression Vector.................................................................................................................. 16 Methods and Materials ............................................................................................................................ 16 Construction of the Expression Vectors .......................................................................................... 16 Construction of the Expression Strain ............................................................................................ 20 Detection Methods for α-Gal ............................................................................................................ 21 Plate Assays ........................................................................................................................................ 21 Whole Cell Enzyme Assays .............................................................................................................. 21 Results ..................................................................................................................................................... 22 Construction of human α-Gal Expression Vectors: ....................................................................... 22 Construction and evaluation of E. coli expression strain .............................................................. 24 Discussion ............................................................................................................................................... 27 Chapter 3 Site directed mutagenesis of human α-Gal in P. pastoris .......................................................... 28 Overview ................................................................................................................................................. 28 Active Site Mutants ........................................................................................................................... 28 vii Interface Mutations .......................................................................................................................... 33 Third Glycosylation Site Mutants ................................................................................................... 35 Methods and Materials ...........................................................................................................................
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