Clark Nathaniel E, Garman Scott C
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, 01003, USA.
J Mol Biol. 2009 Oct 23;393(2):435-47. doi: 10.1016/j.jmb.2009.08.021. Epub 2009 Aug 14.
alpha-N-acetylgalactosaminidase (alpha-NAGAL; E.C. 3.2.1.49) is a lysosomal exoglycosidase that cleaves terminal alpha-N-acetylgalactosamine residues from glycopeptides and glycolipids. In humans, a deficiency of alpha-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 alpha-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 alpha-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 alpha-NAGAL glycoprotein lead to Schindler disease, we analyzed the effect of disease-causing mutations on the three-dimensional structure.
α-N-乙酰半乳糖胺酶（α-NAGAL；E.C. 3.2.1.49）是一种溶酶体外切糖苷酶，可从糖肽和糖脂中切割末端α-N-乙酰半乳糖胺残基。在人类中，α-NAGAL活性缺乏会导致溶酶体贮积症——辛德勒病和神崎病。为了更好地了解这些疾病中的分子缺陷，我们在重组昆虫细胞表达系统中表达野生型和糖基化缺陷型糖蛋白后，测定了人α-NAGAL的晶体结构。我们测量了纯化的野生型和突变型酶的酶学参数，确定了它们的酶活性相当。为了研究人α-NAGAL酶的结合特异性和催化机制，我们测定了三种晶体复合物，其活性位点结合了不同的催化产物。为了更好地理解α-NAGAL糖蛋白中的个体缺陷如何导致辛德勒病，我们分析了致病突变对三维结构的影响。
