Program: Molecular & Cellular Biology
Advisor: Scott C. Garman, Ph.D.
Education: University of Massachusetts, BS, Cum Laude, Biochemistry & Molecular Biology, 2006-2010
Investigate the molecular basis of the inherited metabolic disease known as Fabry disease
Structural Studies of Human Acid Ceramidase
Human acid ceramidase (AC) is deficient in the lysosomal storage disorder known as Farber disease. Overexpression of the enzyme also promotes tumor growth in some prostate cancers. Treatments for Farber disease and other diseases associated with defects in AC have been hampered by a lack of structural information about the enzyme. Our research goal is to determine the structure of recombinant human acid ceramidase, an enzyme currently under clinical development for the treatment of Farber disease. The structure will allow an understanding of the mechanism of AC and facilitate the design of specific AC inhibitors, which are currently unavailable, that might lead to tumor suppression in prostate cancer. Crystals of the fully glycosylated enzyme were obtained but are not ideal for structural studies, because they require months to grow. The AC enzyme was enzymatically deglycosylated to screen for new crystal forms. Deglycosylated AC proved to be a much better reagent for crystallography, leading to reproducible crystals. X-ray diffraction studies on the new crystals will be tested in the near future.
Biophysical Studies of a Monomeric Form of Human Alpha Galactosidase A
The human alpha-galactosidase A (GLA) enzyme is deficient in Fabry disease, and patients suffer from accumulation of undegraded substrates in organs including the skin, heart, eyes, and kidneys. Fabry disease can be treated by enzyme replacement therapy, an FDA-approved approach where recombinant enzyme is injected into patients, and the recombinant enzyme replaces the patient’s missing enzyme. Despite the overall success of enzyme replacement therapy for treating Fabry disease, the kidneys of patients on the treatment can accumulate undegraded substrates and thus lose function. We hypothesize that a smaller variant of alpha-galactosidase A would be able to pass through the semipermeable basement membrane in the kidney, and thus reduce accumulation of substrates in the podocyte cells of the kidney. Our goal is to design a smaller recombinant enzyme that better pass through the filtration barrier in the kidneys. Starting from the wild-type dimeric alpha-galactosidase A, we engineered a monomeric version of the enzyme. We successfully expressed the monomeric alpha-galactosidase A in a yeast expression system, but the resulting protein was hyperglycosylated and produced in low yield. We then switched to a baculovirus expression system to produce protein in insect cells. The baculovirus system produced a uniformly glycosylated monomeric alpha-galactosidase A at yields more than 50-fold higher than the yeast system. A new purification scheme for the monomeric alpha-galactosidase A was developed, using large-scale dialysis followed by nickel and ion exchange chromatography to produce pure protein more consistently. The monomeric alpha-galactosidase A shows less enzyme activity compared to the dimeric wild-type enzyme. We then pursued protein design strategies to increase the activity of this monomeric enzyme. Using homologous structures of alpha-galactosidase A as design templates, we produced twelve additional variants and tested their enzymatic activity. However, the additional variants all showed decreased activity. Further engineering efforts are now underway to increase the stability and activity of our engineered monomeric form of alpha-galactosidase A. We will also test if the smaller enzyme is delivered more efficiently to podocyte cells compared to the wild-type alpha-galactosidase A.