ABSTRACT
BACKGROUND: Recently, enzyme enhancement therapy (EET) for Pompe disease involving imino sugars, which act as potential inhibitors of acid alpha-glucosidases in vitro, to improve the stability and/or transportation of mutant acid alpha-glucosidases in cells was studied and attracted interest. However, the mechanism underlying the molecular interaction between the imino sugars and the enzyme has not been clarified yet. METHODS: We examined the inhibitory and binding effects of four imino sugars on a recombinant human acid alpha-glucosidase, alglucosidase alfa, by means of inhibition assaying and isothermal titration calorimetry (ITC). Furthermore, we built structural models of complexes of the catalytic domain of the enzyme with the imino sugars bound to its active site by homology modeling, and examined the molecular interaction between them. RESULTS: All of the imino sugars examined exhibited a competitive inhibitory action against the enzyme, 1-deoxynojirimycin (DNJ) exhibiting the strongest action among them. ITC revealed that one compound molecule binds to one enzyme molecule and that DNJ most strongly binds to the enzyme among them. Structural analysis revealed that the active site of the enzyme is almost completely occupied by DNJ. CONCLUSION: These biochemical and structural analyses increased our understanding of the molecular interaction between a human acid alpha-glucosidase and imino sugars.
Subject(s)
Glycogen Storage Disease Type II/enzymology , Imino Sugars/metabolism , alpha-Glucosidases/metabolism , 1-Deoxynojirimycin/chemistry , 1-Deoxynojirimycin/metabolism , 1-Deoxynojirimycin/pharmacology , Binding Sites , Catalytic Domain , Drug Interactions , Glycoside Hydrolase Inhibitors , Humans , Imino Sugars/chemistry , Imino Sugars/pharmacology , Models, Molecular , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , ThermodynamicsABSTRACT
We constructed structural models of the catalytic domain and the surrounding region of human wild-type acid alpha-glucosidase and the enzyme with amino acid substitutions by means of homology modeling, and examined whether the amino acid replacements caused structural and biochemical changes in the enzyme proteins. Missense mutations including p.R600C, p.S619R and p.R437C are predicted to cause apparent structural changes. Nonsense mutation of p.C103X terminates the translation of acid alpha-glucosidase halfway through its biosynthesis and is deduced not to allow formation of the active site pocket. The mutant proteins resulting from these missense and nonsense mutations found in patients with Pompe disease are predictably unstable and degraded quickly in cells. The structural change caused by p.G576S is predicted to be small, and cells from a subject homozygous for this amino acid substitution exhibited 15 and 11% of the normal enzyme activity levels for an artificial substrate and glycogen, respectively, and corresponding amounts of the enzyme protein on Western blotting. No accumulation of glycogen was found in organs including skeletal muscle in the subject, and thus the residual enzyme activity could protect cells from glycogen storage. On the other hand, p.E689K, which is known as a neutral polymorphism, little affected the three-dimensional structure of acid alpha-glucosidase. Structural study on a mutant acid alpha-glucosidase in silico combined with biochemical investigation is useful for understanding the molecular pathology of Pompe disease.
