The 1.9 Astroms Structure of Human α-N-Acetylgalactosaminidase: The Molecular Basis of Schindler and Kanzaki Diseases

?-N-acetylgalactosaminidase (?-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal ?-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of ?-NAGAL activity results in the lysosomal storage disorders Schindler disease and Kanzaki disease. To better understand the molecular defects in the diseases, we determined the crystal structure of human ?-NAGAL after expressing wild-type and glycosylation-deficient glycoproteins in recombinant insect cell expression systems. We measured the enzymatic parameters of our purified wild-type and mutant enzymes, establishing their enzymatic equivalence. To investigate the binding specificity and catalytic mechanism of the human ?-NAGAL enzyme, we determined three crystallographic complexes with different catalytic products bound in the active site of the enzyme. To better understand how individual defects in the ?-NAGAL glycoprotein lead to Schindler disease, we analyzed the effect of disease-causing mutations on the three-dimensional structure.