Potential Modifications to Enzyme Replacement Therapy in Anderson-Fabry Disease

Mutations in the GLA gene that encodes the lysosomal enzyme α-galactosidase A (αGal) result in the sphingolipidoses named Fabry disease. This enzymatic defect is inherited as an X-linked recessive disorder and is associated with a progressive deposition of glycosphingolipids, including globotriaosylceramide (GB3), galabioasylceramide, and blood group B substance in the cell. In affected males, and in some females, this leads to early death due to occlusive disease of the heart, kidney, and brain. This disease is currently treated by infusions of αGal, prolonging patients’ lives but producing antibodies against the enzyme reducing the treatment efficacy. Treatment also causes numerous and sometimes life threatening infusion related adverse reactions, including anaphylactic shock, and even death in rare occasions. Here we propose two potential improvements to the current therapeutic practices which would allow for more effective enzyme therapies. The first is constructing and analyzing potentially more active carboxyl-terminal deletions of αGal and the second focuses on targeting of αGal to the very high uptake scavenger receptor (SR) for improved transport to the lysosome

Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu

Carboxyl-Terminal Truncations Alter the Activity of the Human α-Galactosidase A

<div><p>Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the human lysosomal enzyme, α-galactosidase A (αGal), leading to strokes, myocardial infarctions, and terminal renal failure, often leading to death in the fourth or fifth decade of life. The enzyme is responsible for the hydrolysis of terminal α-galactoside linkages in various glycolipids. Enzyme replacement therapy (ERT) has been approved for the treatment of Fabry disease, but adverse reactions, including immune reactions, make it desirable to generate improved methods for ERT. One approach to circumvent these adverse reactions is the development of derivatives of the enzyme with more activity per mg. It was previously reported that carboxyl-terminal deletions of 2 to 10 amino acids led to increased activity of about 2 to 6-fold. However, this data was qualitative or semi-quantitative and relied on comparison of the amounts of mRNA present in Northern blots with αGal enzyme activity using a transient expression system in COS-1 cells. Here we follow up on this report by constructing and purifying mutant enzymes with deletions of 2, 4, 6, 8, and 10 C-terminal amino acids (Δ2, Δ4, Δ6, Δ8, Δ10) for unambiguous quantitative enzyme assays. The results reported here show that the <i>k</i>_<i>cat</i>/<i>K</i>_<i>m</i> approximately doubles with deletions of 2, 4, 6 and 10 amino acids (0.8 to 1.7-fold effect) while a deletion of 8 amino acids decreases the <i>k</i>_<i>cat</i>/<i>K</i>_<i>m</i> (7.2-fold effect). These results indicate that the mutated enzymes with increased activity constructed here would be expected to have a greater therapeutic effect on a per mg basis, and could therefore reduce the likelihood of adverse infusion related reactions in Fabry patients receiving ERT treatment. These results also illustrate the principle that <i>in vitro</i> mutagenesis can be used to generate αGal derivatives with improved enzyme activity.</p></div

Literature Values for <i>K</i>_<i>m</i> and <i>V</i>_<i>max</i> for the WT Human <i>α</i>Gal.

<p>Note. The values given are for the human enzyme purified directly from human tissues or from the indicated recombinant sources. Replagal is produced in human foreskin fibroblasts and Fabrazyme is produced in CHO cells. The average from these literature values are 2.6 ± 0.9mM (<i>K</i>_<i>m</i>) and 3.2 ± 1.1mmole/hr/mg (<i>V</i>_<i>max</i>). NA: not available. MUG was used as the substrate to determine the <i>K</i>_<i>m</i> and <i>V</i>_<i>max</i> values.</p><p>Literature Values for <i>K</i>_<i>m</i> and <i>V</i>_<i>max</i> for the WT Human <i>α</i>Gal.</p

The C-termini of human and coffee <i>α</i>galactosidase.

<p>The crystal structure of human <i>α</i>Gal and a predicted model of the coffee homolog were superimposed. Underlined terminal residues, (MSLKDLL) in humans and (Q) in the coffee bean enzyme, indicate amino acids that could not be modeled due to conformational disorder. The terminal amino acid of the coffee enzyme (glutamine, Q) aligns with (threonine, T) in the human enzyme and is located 9 amino acids (MQMSLKDLL) from the C-terminus of the human enzyme.</p

Purification Table for WT <i>α</i>Gal Expressed in <i>P. pastoris</i>.

<p>Note. 5 mM MUG was used as the substrate for enzyme assay.</p><p>Purification Table for WT <i>α</i>Gal Expressed in <i>P. pastoris</i>.</p

Thermostability profiles of WT and mutant αGal.

<p>Stability of recombinant WT and Δ2 to Δ10 mutant <i>α</i>Gal at 30°C (a), 40°C (b), and 50°C (c) at pH 5.5 as monitored by fluorescent enzyme assay. Initial activities ranged from approximately 300 to 1,900 units/mL for all enzymes assayed. % Activity is normalized against activity at t = 0 mins. Data points for (a) and (c) are the mean of a triplicate measurement with error bars equivalent to ± 1 standard deviation. Data points for (b) are the results of a single measurement. MUG was used as the substrate for enzyme assay.</p

Literature Values of <i>K</i>_<i>m</i>, <i>k</i>_<i>cat</i>, and the specificity constant (<i>k</i>_<i>cat</i>/<i>K</i>_<i>m</i>) for Glycosyl Hydrolase Family 27 <i>α</i>Gal Enzymes.

<p>Note. PNP<i>α</i>Gal substrate was used to calculate kinetic values. Family 27 enzymes include the human <i>α</i>Gal and related enzymes in the CAZy database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118341#pone.0118341.ref102" target="_blank">102</a>] that are most closely related as indicated by BLAST analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118341#pone.0118341.ref094" target="_blank">94</a>]. *<i>k</i>_<i>cat</i> values for <i>S. erythraea</i> and <i>C. josuiI</i> were calculated based on the reported <i>V</i>_<i>max</i>, and molecular weights.</p><p>Literature Values of <i>K</i>_<i>m</i>, <i>k</i>_<i>cat</i>, and the specificity constant (<i>k</i>_<i>cat</i>/<i>K</i>_<i>m</i>) for Glycosyl Hydrolase Family 27 <i>α</i>Gal Enzymes.</p

pH activity curves of WT and mutant αGal.

<p>pH activity curves for WT and Δ2 to Δ10 mutant <i>α</i>Gal. % Activity is normalized against each enzyme’s peak activity. Data points are the mean of a triplicate measurement and error bars are ± 1 standard deviation. MUG was used as the substrate for enzyme assay.</p

Strains and Plasmids.

<p>Strains and Plasmids.</p