Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.

Publication Type:

Journal Article

Authors:

Tomasic, Ivan B; Metcalf, Matthew C; Guce, Abigail I; Clark, Nathaniel E; Garman, Scott C

Source:

J Biol Chem, Volume 285, Issue 28, p.21560-6 (2010)

Keywords:

alpha-Galactosidase, alpha-N-Acetylgalactosaminidase, Amino Acid Sequence, Carbohydrate Metabolism, Carbohydrates, Crystallography, X-Ray, Fabry Disease, Glycoproteins, Humans, Kinetics, Lysosomal Storage Diseases, Lysosomes, Molecular Sequence Data, Protein Folding, Sequence Homology, Amino Acid, Substrate Specificity

Abstract:

<p>The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.22) and alpha-N-acetylgalactosaminidase (alpha-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of alpha- GAL and alpha-NAGAL. The engineered alpha-GAL (which we call alpha-GAL(SA)) retains the antigenicity of alpha-GAL but has acquired the enzymatic specificity of alpha-NAGAL. Conversely, the engineered alpha-NAGAL (which we call alpha-NAGAL(EL)) retains the antigenicity of alpha-NAGAL but has acquired the enzymatic specificity of the alpha-GAL enzyme. Comparison of the crystal structures of the designed enzyme alpha-GAL(SA) to the wild-type enzymes shows that active sites of alpha-GAL(SA) and alpha-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.</p>