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

Abstract

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 k cat/K m 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 k cat/K m (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 in vitro mutagenesis can be used to generate αGal derivatives with improved enzyme activity.

Optimizing human α-galactosidase for treatment of Fabry disease

“…Other approaches for improving the intrinsic limitations of GLA include rational mutagenesis of cysteine residues 49 , active site, dimer interface, and glycosylation sites 50 , deletion of C-terminal sequences 51 , rational modification of alternative enzymes 32 , the use of alternative expression hosts, such as moss 52 and tobacco cells 48 , glycoengineering to improve affinity to the M6P receptor or increase M6P moieties on the enzyme 32,[53][54][55] , and fusion proteins for improved tissue targeting 56,57 . While each of these approaches addresses aspects of intrinsic GLA shortcomings, our approach allows for the comprehensive optimization of multiple properties simultaneously via high-throughput screening of variant libraries under conditions that mimic the path the therapy is exposed to and the natural inactivation strategies it may encounter in the human body upon administration as ERT.…”

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease

“…For example, Qui et al showed that mutants C90S, C174S, and C90S/C174S are enzymatically active, structurally intact, and thermodynamically stable, as measured by circular dichroism and thermal denaturation [ 37 ]. In another study, C-terminal deletions of the GLA enzyme showed not only better enzymatic performance but also an equal thermal stability at 30 °C, 40 °C, and 50 °C for wild-type and C-terminal GLA mutants [ 46 ]. These studies show that researchers in the field are fully aware of the importance of other parameters than only enzymatic activity to check new putative candidates for ERT in FD.…”

“…Generally, this type of mutation is associated with a severe phenotype. However, removal of the first residues has been found to give rise to a protein with superior enzyme activity [ 46 , 47 ].…”

“…Further characterization of the carboxyl-terminal truncations was carried out by Meghdari et al [ 46 ]. They produced (in the methylotrophic yeast Pichia Pastoris ) and purified the mutant enzymes with deletions of 2, 4, 6, 8, and 10 C-terminal amino acids (Δ2, Δ4, Δ6, Δ8, Δ10, respectively) to perform quantitative enzyme assays.…”

Nanotechnology‐based approaches for treating lysosomal storage disorders, a focus on Fabry disease

“…Production of GLA enzyme, was tested in different systems including microbial cell factories such as Escherichia coli and the psychrophilic bacterium Pseudoalteromonas haloplanktis (Ferrer‐Miralles et al, 2011; Rodríguez‐Carmona et al, 2015; U. Unzueta et al, 2015) but since the glycosylation pattern of the protein is very relevant to its final activity, we finally choose to use HEK293F (human embryonic kidney cells) and CHO DG44 cells, widely used in the production of human recombinant proteins. To facilitate the purification of recombinantly produced GLA, a histidine (His) tag (rendering GLA‐His protein) or a cmycHis tag (rendering GLAcmycHis protein) were included in the C‐terminus of the protein, a region of the GLA protein could be modified to increase its enzymatic activity (Meghdari, Gao, Abdullahi, Stokes, & Calhoun, 2015). For the former, GLA‐His was produced in HEK‐293 cells by transfection and transient gene expression while the GLAcmycHis was obtained from CHO cells stably transfected with the GLA coding DNA (Corchero et al, 2011).…”