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Author ORCID Identifier


Open Access Dissertation

Document Type


Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded


Month Degree Awarded


First Advisor

Scott Garman

Second Advisor

Peter Chien

Third Advisor

Jeanne Hardy

Fourth Advisor

Daniel Hebert

Subject Categories

Biochemistry | Structural Biology


Fabry disease is an X-linked lysosomal storage disorder that affects

approximately 1 in 40,000 males in its classical form and as many as 1:4,600 in its

late-onset form [1]. The disease is caused by mutations in the gene encoding α-

galactosidase (α-GAL), which results in deficient levels of α-GAL activity in the

lysosomes of patients [2, 3]. This lack of enzymatic activity causes macromolecular

substrates to accumulate in tissues, and can result in a wide range of symptoms such

as impaired renal and cardiac function [4]. The severity of disease is linked to the

amount of residual enzyme activity [5, 6]. Mutations resulting in little to no residual

activity lead to the more severe classical form of the disease, whereas those that

retain a fraction of wild-type enzyme levels lead to the less severe late-onset form of

the disease. The FDA has approved both enzyme replacement therapy (ERT) and

pharmacological chaperone therapy (PCT) for the treatment of Fabry disease.

ERT involves the intravenous injection of recombinant α-GAL. The injected

enzyme is delivered to the lysosome through the binding of extracellular mannose-

6-phosphate receptors. ERT has been shown to clear accumulated substrate in the

majority of tissue types, and has shown to slow the impairment of organ function

typically associated with the disease [7, 8]. Despite being broadly efficacious for the

treatment of both forms of Fabry disease, additional treatment options were


PCT recently received approval for the treatment of a select number of Fabry

patients. The treatment involves the oral administration of Galafold™, a small

molecule also known as 1-deoxygalactonojirimycin (DGJ). DGJ is a potent competitive

inhibitor of α-GAL, and acts by stabilizing mutant forms of α-GAL as they traffic to

the lysosome [9, 10]. Only a portion of Fabry patients are approved to receive this

treatment [11]. These patients possess one of the 348 genetic mutations for which this

treatment has been found applicable. The majority of these patients have the late-onset

form of the disease and possess some level of residual α-GAL activity. Despite

the success of PCT and ERT at treating a broad number of patients there still exists a

number of shortcomings associated with either treatment.

One such shortcoming is the immunogenicity associated with the

recombinant ERT enzymes. 88% of patients receiving ERT develop immune

responses including both IgG and IgE based reactions [12-14]. It is our belief that the

immune response is present because classical Fabry patients produce no correctly

folded α-GAL, and when correctly folded α-GAL is presented to the immune system

it is treated as foreign. This results in immune response and is likely to trigger the

formation of neutralizing antibodies. In order to bypass this issue, we interconverted

the active sites of α-GAL and a highly homologous human enzyme, α-NAGAL. The

engineered enzymes possess lower catalytic efficiency than the wild-type

counterparts, but have acquired the selectivity of their counterparts. We confirmed

this through enzymatic characterization and through x-ray crystallography. Most

importantly we showed that enzymes retain their native antigenicity. By

engineering novel functionality into previously existing protein scaffolds we have

highlighted a rational approach to engineering less immunogenic therapeutics.

In addition to possessing undesirable immunogenic properties, the efficiency

of ERT in the clearance of substrate in podocyte cells has come into question [15, 16]. These cells play a critical role in the function of the glomerular filtration barrier and

ultimately kidney function. Due to the function of these cells, they form the third

layer of filtration as well as performing vital role in the structure of glomerular

capillaries, they are naturally a problematic cell type for serum-circulated ERT

molecules to reach. Permeability of this barrier has been shown to be dependent on

charge and molecular diameter, with permeability having an inverse relationship

with molecular diameter. By engineering mutations that disrupt the dimer interface

of α-GAL we have successfully expressed and purified a monomeric form of the

enzyme, which possesses slightly lower than wild-type levels of activity and

stability. The monomeric α-GAL may provide an excellent point of entry towards

engineering a more potent ERT molecule for the treatment of renal variants of Fabry


While investigating the selectivity and affinity of α-Gal and α-NAGAL towards

galactose and α-N-acetylgalactosamine analogs, we observed that at concentrations

similar to those used in clinical trials DGJ significantly inhibits human β-GAL. This

observation led us to further investigate the effect of α-linked functional groups

attached to the position corresponding to C1 in a galactose scaffold. In DGJ and the

2-acetamido variant, DGJNAc, this position is occupied by a hydrogen. We utilized

inhibition assays and x-ray crystallography to probe the effect of certain functional

groups at this position on specificity and affinity towards α-GAL or α-NAGAL. We

determined that these groups provided little to no change in affinity, but

provided increased specificity towards α-selecting active sites. These findings coupled

with previous studies performed by members of the Garman lab provide a clear set

of guidelines towards developing ligands specific towards either α-GAL α-NAGAL.