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Carboxyl-terminal truncations alter the activity of the human ?-galactosidase A.

ABSTRACT: Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the human lysosomal enzyme, ?-galactosidase A (?Gal), 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 ?-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 ?Gal 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 (?2, ?4, ?6, ?8, ?10) for unambiguous quantitative enzyme assays. The results reported here show that the kcat/Km 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 kcat/Km (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 ?Gal derivatives with improved enzyme activity.

SUBMITTER: Meghdari M

PROVIDER: S-EPMC4342250 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Carboxyl-terminal truncations alter the activity of the human α-galactosidase A.

Meghdari Mariam M Gao Nicholas N Abdullahi Abass A Stokes Erin E Calhoun David H DH

PloS one 20150226 2

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, includi ...[more]

PMID: 25719393

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Project description:In the cell, misfolded proteins are processed by molecular chaperone-mediated refolding or through ubiquitin-mediated proteosome system. Dysregulation of these mechanisms facilitates the aggregation of misfolded proteins and forms aggresomes in the juxta nuclear position of the cell which are removed by lysosome-mediated autophagy pathway in the subsequent cell division. Accumulation of misfolded proteins in the cell is hallmark of several neurological disorders and other diseases including cancer. However, the exact mechanism of aggresome formation and clearance is not thoroughly understood. Reports have shown that several proteins including p300, p53, TAU, ?-synuclein, SOD, etc. contain intrinsically disordered region (IDR) which has the tendency to form aggresome. To study the nature of aggresome formation and stability of the aggresome, we have chosen Twist1 as a model protein since it has IDR regions. Twist1 is a bHLH transcription factor which plays a major role in epithelial mesenchymal transition (EMT) and shown to interact with HAT domain of p300 and p53. In the present study, we generated several deletion mutants of human Twist1 with different fluorescent tags and delineated the regions responsible for aggresome formation. The Twist1 protein contains two NLS motifs at the N-terminal region. We showed that the deletions of regions spanning the amino acids 30-46 (Twist1?30-46) which lacks the first NLS motif form larger and intense aggregates while the deletion of residues from 47 to 100 (Twist1?47-100) which lacks the second NLS motif generates smaller and less intense aggregates in the juxta nuclear position. This suggests that both the NLS motifs are needed for the proper nuclear localization of Twist1. The aggresome formation of the Twist1 deletion mutants was confirmed by counterstaining with known aggresome markers: Vimentin, HDAC6, and gamma tubulin and further validated by MG-132 treatment. In addition, it was found that the aggresomes generated by the Twist1?30-46 construct are more stable than the aggresome produced by the Twist1?47-100 construct as well as the wild-type Twist1 protein. Taken together, our data provide an important understanding on the role of IDR regions on the formation and stability of aggresomes.

Dataset Information

Carboxyl-terminal truncations alter the activity of the human ?-galactosidase A.

Publications

Carboxyl-terminal truncations alter the activity of the human α-galactosidase A.

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