Garman lab: Interconversion of lysosomal enzyme specificities

Lysosomal storage diseaseLysosomal storage disease

Lysosomal storage disorders are inherited metabolic diseases characterized by an accumulation of undigested various toxic materials. There are nearly 50 diseases and the two examples shown here are Fabry and Schindler disease. Fabry disease, which occurs between early childhood and adolescence, is characterized by the lack of the enzyme alpha galactosidase (α-Gal). Schindler disease can occur in infancy or in adulthood and is characterized by the lack on the enzyme alpha N-acetylgalactosaminidase (α-NaGal). There are currently no cures for lysosomal storage disorders however enzyme replacement therapy is a treatment option. The basic principle of enzyme replacement therapy is to over express the enzyme of interest heterologously, in this case α-Gal α-NaGal, in a cell line and to isolate and purify it from the culture. In enzyme replacement therapy, patients are injected with the enzymes that they lack in the hopes of restoring the enzymatic activity in their cells.

Immune ResponseImmune Response

Individuals suffering from Fabry disease cannot produce the Alpha-Gal protein that is necessary for breaking down Galactose. The usual treatment for this is giving the patient doses of the protein, but this poses a problem. Since the body does not produce the protein, an immune response ranging from severe anaphylaxis to mild discomfort can occur when the patient is given the protein. The body does however produce Alpha NAGAL, a protein with a similar active site and function as Alpha Gal. Altering the active site of Alpha NAGAL to match that of Alpha Gal allows doctors to administer a protein that serves the function of Alpha Gal but has the antigenicity of Alpha NAGAL, which means the body will recognize the protein and not elicit an immune response.

Enzymatic activityEnzymatic activity

α-Gal and α-NaGal have relatively identical active sites, which are conserved with the exception of alanine, serine, glutamate and leucine which are positioned differently. The two enzymes have the same folds and both function by cleaving glycosydic bonds however have different substrate specificities. The differences in substrate specificity occur because α-NaGal has a larger binding pocket thus interacting with larger molecules but smaller residues.

Galactose vs. N-acetyl-galactosamine

Structures shown on this pageStructures shown on this page

3H54: alpha-NAGAL with GalNAc

3HG5: alpha-GAL with Gal

3LX9: alpha-GAL(SA) in complex with GalNAc

3LXA: alpha-GAL(SA) in complex with Galactose

3LXC: alpha-GAL(SA) in presence of Glycerol

Figure 1 Panel B () Panel C (show ) Figure 2: Structure of alpha-GAL(SA) The substitution of Glu 203 and Leu 206 with Serine and alanine reduces the size of the side chain in E203S substitution. The hydrogen bonding of Asp 170 to Tyr 134 or Tyr 207 is altered, thus opening the active site to resemble that of alpha NAGAL. These contribute to the reduced catalytic effect of alpha GAL SA. : in complex with GalNAc Crystal structure of alpha-GAL(SA) bound to GalNAc. Alpha-GAL(SA) active site residues are shown in yellow and the product, GalNAc, is shown in gray. The blue frame around GalNAc represents its electron density. Red and blue spheres in the figure represent oxygen and nitrogen, respectively. : in complex with GalNAc : Superposition with alpha-NAGAL bound to GalNAc Superposition of structures: alpha-GAL(SA) bound to GALNAc, in yellow, and alpha-NAGAL bound to GALNAc, in dark blue. GALNAc product of alpha-GAL(SA) is shown in light brown and GALNAc. product of alpha-NAGAL is shown in light blue. The two product structures are oriented slightly at different angles, but no difference in binding was found.

spinqualitylabelsall models Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image