Human acid β-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites

Osiecki-Newman, Karen; Fabbro, Diane; Legler, Günter; Desnick, Robert J.; Grabowski, Gregory A.

doi:10.1016/0167-4838(87)90128-2

Cited by 51 publications

(37 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Estimation of Enzyme Activity-Acid ␤-glucosidase activities in the presence of phosphatidylserine and saposins were estimated by quantitating the fluorescent intensity of liberated 4-methylumbelliferone from the fluorogenic substrate, 4-methylumbelliferyl-␤-D-glucopyranoside (15,21). The usual reaction mixture (200 l) contained 2 nM acid ␤-glucosidase, phosphatidylserine (0.4 g/ml), 4-methylumbelliferyl-␤-D-glucopyranoside (4 mM), 0.1 M sodium acetate, pH 4.7, and the indicated amounts of saposin (15).…”

Section: Materials-thementioning

confidence: 99%

Functional Organization of Saposin C

Qin

Sun

et al. 1996

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Saposin C is an essential co-factor for the hydrolysis of glucosylceramide by acid ␤-glucosidase in mammals. In addition, prosaposin promotes neurite outgrowth in vitro via sequences in saposin C. The regional organization of these neurotrophic and activation properties of saposin C was elucidated using recombinant or chemically synthesized saposin Cs from various regions of the molecule. Unreduced and reduced proteins were analyzed by electrospray-mass spectrometry to establish the complement of disulfide bonds in selected saposin Cs. Using saposin B as a unreactive backbone, chimeric saposins containing various length segments of saposin B and C localized the neurotrophic and acid ␤-glucosidase activation properties to the carboxyl-and NH 2 -terminal 50% of saposin C, respectively. The peptide spanning residues 22-31 had neurotrophic effects. Molecular modeling and site-directed mutagenesis localized the activation properties of saposin C to the region spanning residues 47-62. Secondary structure was needed for retention of this property. Single substitutions of R and S at the conserved cysteines at 47 or 78 diminished but did not obliterate the activation properties. These results indicate the segregation of neurotrophic and activation properties of saposin C to two different faces of the molecule and suggest a topographic sequestration of the activation region of prosaposin for protection of the cell from adverse hydrolytic activity of acid ␤-glucosidase.

show abstract

Section: Materials-thementioning

confidence: 99%

Functional Organization of Saposin C

Qin

Sun

et al. 1996

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…36 mutations have so far been reported in the GlcCerase gene, including point mutations, insertional mutations, deletions and splicing mutations (Beutler, 1993;Mistry and Cox, 1993;Horowitz and Zimran, 1994). All of these mutations result in a large, but comparable, decrease in GlcCerase activity when measured in vitro using a variety of synthetic substrates (Hultberg and Ockerman, 1972;Dinur et al, 1984;Osiecki-Newman et al, 1987). Thus no correlation was observed in vitro between the V.,ax of GlcCerase towards 4-methylumbelliferyl /-D-gluoco- [Futerman and Pagano (1991) Biochem.…”

Section: Introductionmentioning

confidence: 82%

Analysis of glucocerebrosidase activity using N-(1-[14C]hexanoyl)-d-erythro-glucosylsphingosine demonstrates a correlation between levels of residual enzyme activity and the type of Gaucher disease

Meivar‐Levy

Horowitz

Futerman

1994

Biochemical Journal

View full text Add to dashboard Cite

Glucosylceramide, a degradation product of complex glycosphingolipids, is hydrolysed in lysosomes by glucocerebrosidase (GlcCerase). Mutations in the human GlcCerase gene cause a reduction in GlcCerase activity and accumulation of glucosylceramide, which results in the onset of Gaucher disease, the most common lysosomal storage disease. Significant clinical heterogeneity is observed in Gaucher disease, with three main types known, but no clear correlation has been reported between the different types and levels of residual GlcCerase activity. We now demonstrate that a correlation exists by using a radioactive, short-acyl chain substrate, N-(1-[14C]hexanoyl)-D-erythro-glucosylsphingosine ([14C]hexanoyl-GlcCer). This substrate rapidly transferred into biological membranes in the absence of detergent [Futerman and Pagano (1991) Biochem. J. 280, 295-302] and was hydrolyzed to N-(1-[14C]hexanoyl)-D-erythro-sphingosine ([14C]hexanoyl-Cer) both in vitro and in situ, with an acid pH optimum. A strict correlation was observed between levels of [14C]hexanoyl-GlcCer hydrolysis and Gaucher type in human skin fibroblasts. The mean residual activity measured in vitro for 3 h incubation in type 1 Gaucher fibroblasts (the mild form of the disease) was 46.3 +/- 4.6 nmol of [14C]hexanoyl-Cer formed per mg protein (n = 9), and in type 2 and 3 fibroblasts (the neuronopathic forms of the disease) was 19.6 +/- 6.5 (n = 9). A similar correlation was observed when activity was measured in situ, suggesting that the clinical severity of a lysosomal storage disease is related to levels of residual enzyme activity.

show abstract

“…These regions of greatest sequence identity in Fig. 3 may correspond to structural domains (30) within glucocerebrosidase. Therefore, with three different criteria, it appears that-the carboxyl-terminal portion of glucocerebrosidase is more rigidly constrained than the amino-terminal segment if enzymatic activity is to be retained.…”

Section: Resultsmentioning

confidence: 99%

Comparison of the chromosomal localization of murine and human glucocerebrosidase genes and of the deduced amino acid sequences.

O'Neill¹,

Tokoro²,

Kozak³

et al. 1989

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

View full text Add to dashboard Cite

To study structure-function relationships and molecular evolution, we determined the nucleotide sequence and chromosomal location of the gene encoding murine glucocerebrosidase (glucosylceramidase; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45). In the protein coding region of the murine cDNA, the nucleotide sequence and the corresponding deduced amino acid sequences were 82% and 86% identical to the respective human sequences. AU five amino acids presently known to be essential for normal enzymatic activity were conserved between mouse and man. The murine enzyme had a single deletion relative to the human enzyme at amino acid number 273. One ATG translation initiation signal was present in the mouse sequence in contrast to the human sequence, where two start codons have been reported. Nudeotide sequencing of a clone derived from murine genomic DNA revealed that the murine signal for translation initiation was located in exon 2. The locations of all 10 introns were conserved among mouse and man. We mapped the genetic locus for glucocerebrosidase to mouse chromosome 3, at a position 7.6 ± 3.2 centimorgans from the locus for the (3 subunit of nerve growth factor. Comparison of linkage relationships in the human and murine genome indicates that these closely linked mouse genes are also syntenic on human chromosome 1 but in positions that span the centromere.In the lysosomes of normal individuals, acid /-glucocerebrosidase (glucosylceramidase; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2

show abstract

Human acid β-glucosidase: use of inhibitors, alternative substrates and amphiphiles to investigate the properties of the normal and Gaucher disease active sites

Cited by 51 publications

References 30 publications

Functional Organization of Saposin C

Functional Organization of Saposin C

Analysis of glucocerebrosidase activity using N-(1-[14C]hexanoyl)-d-erythro-glucosylsphingosine demonstrates a correlation between levels of residual enzyme activity and the type of Gaucher disease

Comparison of the chromosomal localization of murine and human glucocerebrosidase genes and of the deduced amino acid sequences.

Contact Info

Product

Resources

About