title: 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 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 αGal derivatives with improved enzyme activity.
authors.affiliation: Chemistry Dept., City College of New York, New York, NY, USA.
authors.forename: Mariam
authors.initials: M
authors.lastname: Meghdari
authors.affiliation: Chemistry Dept., City College of New York, New York, NY, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Gao
authors.affiliation: Biology & Medical Lab Technology, Bronx Community College, Bronx, NY, USA.
authors.forename: Abass
authors.initials: A
authors.lastname: Abdullahi
authors.affiliation: Chemistry Dept., City College of New York, New York, NY, USA.
authors.forename: Erin
authors.initials: E
authors.lastname: Stokes
authors.affiliation: Chemistry Dept., City College of New York, New York, NY, USA.
authors.forename: David H
authors.initials: DH
authors.lastname: Calhoun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D006801:Humans; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D009154:Mutation; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D000519:alpha-Galactosidase