Boris Brumshtein

Characterization of gene-activated human acid-β-glucosidase: Crystal structure, glycan composition, and internalization into macrophages

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-β-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-ActivatedTM human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid–base hydrolysis. Kinetic parameters (Km and Vmax) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

Crystal Structures of Complexes of N-Butyl- and N-Nonyl-Deoxynojirimycin Bound to Acid β-Glucosidase INSIGHTS INTO THE MECHANISM OF CHEMICAL CHAPERONE ACTION IN GAUCHER DISEASE

Gaucher disease is caused by mutations in the gene encoding acid β-glucosidase (GlcCerase), resulting in glucosylceramide (GlcCer) accumulation. The only currently available orally administered treatment for Gaucher disease is N-butyl-deoxynojirimycin (Zavesca™, NB-DNJ), which partially inhibits GlcCer synthesis, thus reducing levels of GlcCer accumulation. NB-DNJ also acts as a chemical chaperone for GlcCerase, although at a different concentration than that required to completely inhibit GlcCer synthesis. We now report the crystal structures, at 2Å resolution, of complexes of NB-DNJ and N-nonyl-deoxynojirimycin (NN-DNJ) with recombinant human GlcCerase, expressed in cultured plant cells. Both inhibitors bind at the active site of GlcCerase, with the imino sugar moiety making hydrogen bonds to side chains of active site residues. The alkyl chains of NB-DNJ and NN-DNJ are oriented toward the entrance of the active site where they undergo hydrophobic interactions. Based on these structures, we make a number of predictions concerning (i) involvement of loops adjacent to the active site in the catalytic process, (ii) the nature of nucleophilic attack by Glu-340, and (iii) the role of a conserved water molecule located in a solvent cavity adjacent to the active site. Together, these results have significance for understanding the mechanism of action of GlcCerase and the mode of GlcCerase chaperoning by imino sugars.

6-Amino-6-deoxy-5,6-di-N-(N′-octyliminomethylidene)nojirimycin: Synthesis, Biological Evaluation, and Crystal Structure in Complex with Acid β-Glucosidase

6‐Amino‐6‐deoxy‐5,6‐di‐N‐(N′‐octyliminomethylidene)nojirimycin, a reducing analogue of N‐nonyl‐1‐deoxynojirimycin, proved to be a potent and very selective inhibitor of β‐glucosidases, including human acid β‐glucosidase. Structural studies of the enzyme–inhibitor complex showed a binding mode in which the anomeric hydroxy group is accommodated in the “wrong” α configuration.

Acid Beta-Glucosidase: Insights from Structural Analysis and Relevance to Gaucher Disease Therapy.

Abstract In mammalian cells, glucosylceramide (GlcCer), the simplest glycosphingolipid, is hydrolyzed by the lysosomal enzyme acid β-glucosidase (GlcCerase). In the human metabolic disorder Gaucher disease, GlcCerase activity is significantly decreased owing to one of approximately 200 mutations in the GlcCerase gene. The most common therapy for Gaucher disease is enzyme replacement therapy (ERT), in which patients are given intravenous injections of recombinant human GlcCerase; the Genzyme product Cerezyme® has been used clinically for more than 15 years and is administered to approximately 4000 patients worldwide. Here we review the crystal structure of Cerezyme® and other recombinant forms of GlcCerase, as well as of their complexes with covalent and non-covalent inhibitors. We also discuss the stability of Cerezyme®, which can be altered by modification of its N-glycan chains with possible implications for improved ERT in Gaucher disease.

Control of the rate of evaporation in protein crystallization by the 'microbatch under oil' method.

A procedure is presented for controlling the rate of evaporation during ‘microbatch under oil’ protein crystallization.