Glucocerebrosidase processing in normal fibroblasts and in fibroblasts from patients with type I, type II, and type III Gaucher disease.

Abstract

Fibroblasts from normal subjects and pa- patients with the different types of Gaucher disease using rabbit antisera and suggested that type I disease might be due to a processing error. In our own studies, using affinity-purified heterosera and monoclonal antibodies, we found only a 63-kDa antigen in normal fibroblasts and fibroblasts from patients with type I and type III Gaucher disease. No band was visualized in type II disease. We suggested that the other bands that had been observed by Ginns et al. represented antigens not related to glucocerebrosidase.We now present the results of pulse-chase experiments demonstrating that glucocerebrosidase processing does occur and that processing is quite normal in six patients with type I and one patient with type III Gaucher disease. However, an unstable precursor is formed in type II disease. MATERIALS AND METHODSCultures. Confluent skin fibroblasts cultured in minimal essential medium (MEM) with 20% fetal bovine serum were used in these investigations. Cell lines GM372, GM1260, and GM877 were obtained from the Human Genetic Mutant Cell Repository (Camden, NJ). Other cell lines were derived from our patients with Gaucher disease. Of the type I patients studied, five are Jewish; one is non-Jewish.Antibodies. The production of affinity-purified antiglucocerebrosidase antibodies in rabbits has been described (4). Antibody and preimmune IgG each were immobilized on cyanogen bromide-activated Sepharose beads (1 mg/g of Sepharose; Pharmacia) using the technique recommended by the manufacturer.Pulse Labeling. To pulse label the cells, supernatant culture medium was removed from confluent cultured fibroblasts, they were rinsed with saline, and the medium was replaced by leucine-free MEM containing 5% fetal bovine serum and 0.5 mCi of [3H]leucine Ci/mmol; 1 Ci = 37 GBq; Amersham). After 30 min the medium was removed and the cells were washed four times with saline followed by addition of MEM with 20% fetal bovine serum. Penicillin and streptomycin were used in all medium preparations. One or two flasks containing cultured fibroblasts were sacrificed immediately after labeling (time = 0), at 2 hr, and at 24 hr. The cells were again washed with saline and removed from the flask into saline with a rubber policeman. The cells were pelleted by centrifugation, dissolved in 0.2-0.3 ml of Tris/NaDodSO4 (final concentration, 0.03 M Tris, pH 6.8/1.5% NaDodSO4), and briefly sonicated. The preparations were placed in boiling water for 3 min and frozen until all time points could be processed together.Immunoprecipitation. Glucocerebrosidase antigen was immunoprecipitated using Sepharose-bound antibody. The thawed cell extract was centrifuged at 12,000 x g for 3 min and diluted 1:10 in 0.05 M Tris (pH 6.8) containing 0.1% Triton X-100, 0.4 M NaCl, and 0.6% ovalbumin. Before treating the cell extract with anti-glucocerebrosidase antibody, it was preabsorbed with beads prepared from preimmune rabbit IgG. Two hundred microliters of beads was added to 2 ml of diluted cell extract and ...

Gaucher Disease

“…Early pulse-chase studies of patient fibroblasts [43,44] indicated that the L444P mutation leads to an unstable protein with incompletely processed carbohydrate chains. This observation was later confirmed by recombinant expression in 3T3 and Sf9 cells [35,42,45].…”

Expression of Functional Human Acid ß-Glucosidase in COS-1 and Spodoptera frugiperda Cells

“…Since only single forms (63 kilodaltons) of normal human acid ß-glu cosidase were expressed in COS-1 or Sf9 cells, alternative expression systems will be required to evaluate putative étiologie single base substitutions for these variants. In con trast, several variants of type 1 Gaucher dis ease have been shown to have abnormal active site function [3,11,13,15,26,29] and unaltered glycosidic processing [3,6,8,33, Bergman and Grabowski, unpublished ob servation]. Expression of DNAs from this group of Gaucher variants in Sf9 cells and evaluation of the expressed acid ß-glucosi dase with probes of active site function should allow for the distinction between artifactual or polymorphic and étiologie base substitutions.…”

“…In cul tured fibroblasts, several variants of types 1, 2 and 3 Gaucher disease have been found to have residual mutant enzymes with appar ently intact active site function [6,26,27], but altered post-translational oligosaccha ride remodelling [6, Bergman and Grabowski, unpublished observation] and intracellu lar stabilities [6,8]. On immunoblotting or by pulse-chase processing studies the causal mutations in these variants give leads to sin gle forms of acid ß-glucosidase (usually 63-64 kilodaltons).…”

Generation of Polyclonal Antibodies Against Recombinant Human Glucocerebrosidase Produced in Escherichia coli

“…Anti-GCR antibodies are important and useful tools to characterize GCR produced in a number of expression systems, including insect cells [9,20], plant cells [18] and mammalian cells [21][22][23]. Anti-GCR antibodies can also be used to discriminate between non-neuropathic and neuropathic cases of this disorder, as demonstrated by Ginns and collaborators [24,25], and to characterize the molecular forms of the enzyme in normal individuals and in patients with Types I, II and III forms of the disease [3,11,[26][27][28][29]. So far, anti-GCR antibodies (both monoclonal and polyclonal) have been produced against purified GCR from human placenta [24,30,31].…”