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

Abstract

The human lysosomal enzymes α-galactosidase (α-GAL, EC 3.2.1.22) and α-N-acetylgalactosaminidase (α-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 α-GAL and α-NAGAL. The engineered α-GAL (which we call α-GAL SA) retains the antigenicity of α-GAL but has acquired the enzymatic specificity of α-NAGAL. Conversely, the engineered α-NAGAL (which we call α-NAGALEL) retains the antigenicity of α-NAGAL but has acquired the enzymatic specificity of the α-GAL enzyme. Comparison of the crystal structures of the designed enzyme α-GALSA to the wild-type enzymes shows that active sites of α-GALSA and α-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. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.