title: The effects of chemically synthesized saposin C on glucosylceramide-β-glucosidase.
abstract: OBJECTIVE
  Saposin C (SAP-C) is an essential activator of glucosylceramide (GlcCer)-β-glucosidase (GCase), the enzyme deficient in Gaucher's disease. In this study, we investigated the effects of chemically synthesized SAP-Cs (synthetic SAP-Cs) on GCase.
  
  
  METHODS
  Enzymatic assays and western blot analyses were carried out to evaluate the effects of two kinds of synthetic SAP-Cs, a non-glycosylated form and a N-glycosylated form bearing a complex type nonasaccharide, on GCase with respect to its activation, stabilization, and protection. Imiglucerase (Cerezyme) was used as the GCase. To mimic physiological conditions, GCase activity was assayed in the presence of 4-nitrobenzo-2-oxa-1,3-diazole-labeled GlcCer-containing liposomes composed of bis(monoacylglycero)phosphate, l-α-phosphatidylcholine, and cholesterol.
  
  
  RESULTS
  GCase activities increased depending on the concentration of synthetic SAP-Cs. SAP-Cs at a concentration of 1μM increased GCase activities significantly, by 14- to 22-fold (non-glycosylated SAP-C: 22.9±0.16; nona-glycosylated SAP-C: 14.9±0.19; without SAP-C: 1.05±0.035pmol/h/ng GCase). These values equaled or surpassed previously published values obtained using recombinant non-glycosylated SAP-C. Both synthetic SAP-Cs were as effective as bovine serum albumin in stabilizing GCase at 37°C. Western blot analysis revealed that synthetic SAP-Cs specifically protected GCase from cathepsin D digestion.
  
  
  CONCLUSIONS
  The results demonstrate that these novel, chemically synthesized SAP-Cs function as activators, stabilizers, and protectors of GCase, suggesting their utility in enzyme replacement therapy in patients with Gaucher's disease.
authors.affiliation: Institute of Glycoscience, Tokai University, Kanagawa 259-1292, Japan; Department of Pathology, Faculty of Medicine, Kinki University, Osaka 589-8511, Japan.
authors.forename: Azusa
authors.initials: A
authors.lastname: Yoneshige
authors.affiliation: Institute of Glycoscience, Tokai University, Kanagawa 259-1292, Japan; Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
authors.forename: Masanaga
authors.initials: M
authors.lastname: Muto
authors.affiliation: Institute of Glycoscience, Tokai University, Kanagawa 259-1292, Japan; Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Institute of Glycoscience, Tokai University, Kanagawa 259-1292, Japan; Institute for Protein Research, Osaka University, Osaka 565-0871, Japan.
authors.forename: Hironobu
authors.initials: H
authors.lastname: Hojo
authors.affiliation: Institute of Glycoscience, Tokai University, Kanagawa 259-1292, Japan; Department of Pediatrics, Kawasaki Medical School, Okayama 701-0192, Japan. Electronic address: matsujun@med.kawasaki-m.ac.jp.
authors.forename: Junko
authors.initials: J
authors.lastname: Matsuda
mesh_terms: D002402:Cathepsin D / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D005776:Gaucher Disease / Q000201:enzymology; D005962:Glucosylceramidase / Q000737:chemistry*; D006801:Humans; D049231:Saposins / Q000138:chemical synthesis*
chemical_list: D049231:Saposins; D002784:Cholesterol; D005962:Glucosylceramidase; D002402:Cathepsin D; C090568:imiglucerase
keywords: Cathepsin D; Enzyme replacement therapy; Gaucher's disease; Imiglucerase; Saposin C