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

Abstract

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.

FIGURE 1.

α-GAL and α-NAGAL structural and biochemical analyses. A, sequence alignment of the α-GAL and α-NAGAL proteins. Active site residues are red, and identities have yellow backgrounds. The two active site residues that differ are boxed. B and C, ribbon diagrams of α-GAL (green) and α-NAGAL (cyan) with attached carbohydrates. Insets show the active sites of α-GAL and α-NAGAL with their catalytic products α-galactose and α-GalNAc, respectively (gray). 11 of the 13 active site residues are conserved between the enzymes, although the overall sequence identity is 46%. The two residues that differ (Glu-203 and Leu-206 in α-GAL; Ser-188 and Ala-191 in α-NAGAL) select for the substituent on the 2-position of the ligand. D, the four purified proteins are shown on a Coomassie-stained SDS gel. α-GAL and α-GAL^SA (with three N-linked glycosylation sites each) run smaller on the SDS gel than α-NAGAL and α-NAGAL^EL (with five N-linked glycosylation sites each). E, Western blots of the four proteins, detected with polyclonal anti-α-GAL (top) and polyclonal anti-α-NAGAL antibodies (bottom). The variant proteins retain the antigenicity of the original proteins.

FIGURE 2.

α-GAL^SA crystal structures. A–C, σ_A-weighted 2F_o − F_c total omit electron density maps of α-GAL^SA calculated in SFCHECK (17). A, GalNAc-soaked crystal contoured at 2.0σ. B, galactose-soaked crystal contoured at 1.8σ. C, glycerol-soaked crystal soaked at 1.0σ. Maps have a cover radius drawn around ligands and/or waters in the active site. Active site residues are labeled in A. D, a superposition of crystal structures of the active sites of α-GAL^SA and α-NAGAL, each with α-GalNAc bound in the active site. When the structures are superimposed by their (β/α)₈ barrels, the ligands superimpose nearly exactly. E, a superposition of crystal structures of the active sites of α-GAL^SA and α-GAL, each with α-galactose bound in the active site. F, a superposition of the four monomers of glycerol-soaked α-GAL^SA, with glycerol bound in the active site. In one of the four structures (green), the glycerol binds in a vertical orientation, and shows differences in Arg-227 and the loop containing Asp-231 (arrows).