Gene mapping and leader polypeptide sequence of human glucocerebrosidase: implications for Gaucher disease.

Ginns, Edward I.; Choudary, Prabhakara V; Tsuji, Shoji; Martin, Brian M.; Stubblefield, Barbara; Sawyer, Jeffrey R.; Hozier, John; Barranger, John A.

doi:10.1073/pnas.82.20.7101

Cited by 105 publications

(44 citation statements)

References 21 publications

(22 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…1 legend for details. (6)(7)(8). The mature enzyme is also glycosylated, however, and we do not know whether sugars are added before or after cleavage of the leader sequence.…”

Section: Discussionmentioning

confidence: 99%

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

Beutler¹,

Kühl²

1986

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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 ...

show abstract

“…1 legend for details. (6)(7)(8). The mature enzyme is also glycosylated, however, and we do not know whether sugars are added before or after cleavage of the leader sequence.…”

Section: Discussionmentioning

confidence: 99%

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

Beutler¹,

Kühl²

1986

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…GBA was mapped to 1q21 (Shafit-Zagardo et al 1981;Devine et al 1982;Ginns et al 1985), and GBA cDNA was first cloned and sequenced from a fibroblast library (Sorge et al 1985). The complete genomic sequences of GBA and psGBA were described some years later (GenBank J03059 and J03060, respectively;Horowitz et al 1989).…”

Section: Unitat De Biologia Evolutiva Universitat Pompeu Fabra 0800mentioning

confidence: 99%

Sequence Variability of a Human Pseudogene

Martínez‐Arias¹

2001

Genome Research

View full text Add to dashboard Cite

We have obtained haplotypes from the autosomal glucocerebrosidase pseudogene (psGBA) for 100 human chromosomes from worldwide populations, as well as for four chimpanzee and four gorilla chromosomes. In humans, in a 5420-nucleotide stretch analyzed, variation comprises 17 substitutions, a 3-bp deletion, and a length polymorphism at a polyadenine tract. The substitution rate on the pseudogene (1.23 ± 0.22 × 10 −9 per nucleotide and year) is within the range of previous estimates considering phylogenetic estimations. Recombination within the pseudogene was recognized, although the low variability of this locus prevented an accurate measure of recombination rates. At least 13% of the psGBA sequence could be attributed to gene conversion from the contiguous GBA gene, whereas the reciprocal event has been shown to lead to Gaucher disease. Human psGBA sequences showed a recent coalescence time (∼200,000 yr ago), and the most ancestral haplotype was found only in Africans; both observations are compatible with the replacement hypothesis of human origins. In a deeper timeframe, phylogenetic analysis showed that the duplication event that created psGBA could be dated at ∼27 million years ago, in agreement with previous estimates.

show abstract

“…The genetic distance (cM) and standard error were calculated as previously described (Buckwalter et al 1991). The locations of the human genes are given in parentheses (Arbiser et al 1988;Ginns et al 1985;Nakai et al 1987;Seigel et al 1984). Fig.…”

mentioning

confidence: 99%