Biosynthesis and maturation of glucocerebrosidase in Gaucher fibroblasts (original) (raw)

1987, European Journal of Biochemistry

The biosynthesis and maturation of glucocerebrosidase were studied in fibroblasts from patients with the neurological and non-neurological forms of Gaucher disease and in control cells. In control fibroblasts the precursor of glucocerebrosidase (62-63 kDa), observed after a short pulse with [35S]methionine, was converted during the chase period to a 66-kDa intermediate form and, finally, to the 59-kDa mature protein. In fibroblasts from patients with the non-neurological phenotype of Gaucher disease (type 1) the same biosynthetic forms were seen as in control fibroblasts. These biosynthetic forms correspond to the three-banded pattern seen in control and Gaucher type 1 fibroblast extracts analysed by the immunoblotting procedure, or after electrophoresis and fluorography of extracts of such fibroblasts cultured for 5 days with [14C]leucine. The 59-kDa protein seen in type 1 fibroblasts was unstable and disappeared after a prolonged chase; this disappearance was not observed when the cells were grown in the presence of leupeptin. In fibroblasts from patients with the neurological forms of Gaucher disease (types 2 and 3) the 62.5-kDa precursor of glucocerebrosidase was present in near-normal amounts after a short pulse, but the 59-kDa form was not detected even when cells were cultured with leupeptin. These results are in accordance with the absence of the 59-kDa band in immunoblots of types 2 and 3 fibroblast extracts. Culturing of type 1, type 2 and type 3 Gaucher fibroblasts in the presence of leupeptin led to an increase in the activity of glucocerebrosidase. In Gaucher disease the membrane-associated lysosomal enzyme glucocerebrosidase is deficient [l , 21, resulting in accumulation of the glycosphingolipid glucosyl ceramide in the lysosomes in cells of the reticuloendothelial system [3]. Based on clinical observations, the disease has been divided into three phenotypes [3, 41: type 1, without neurological involvement: type 2, the acute neurological form; and type 3, the subacute neurological form. In patients glucocerebrosidase has been shown to be deficient in liver [5, 61, spleen [5, 7, 81, brain [9, 101, leukocytes [5, 11-131, cultured skin fibroblasts [34], amniotic fluid cells [15] and urine [16]. Correlation between residual activity or stored lipid and clinical subtype has been inadequate (reviewed in [4, 171). However, a discrimination between the non-neurological and neurological phenotypes can be made by an analysis of the cross-reacting material in fibroblast extracts [18, 191. In fibroblasts from control subjects, three forms of immunoreactive glucocerebrosidase are found [18, 191. Fibroblasts from type 1 patients show the same forms of glucocerebrosidase as those from controls, whereas fibroblasts obtained from type 2 and 3 patients are deficient in the smallest-M, species [18, 191. We suggested that the three Correspondence to A. W. Schram, Inter(sub)facultaire Vakgroep Biochemie Universiteit van Amsterdam, P.O. Box 20151, NL-1000-HD Amsterdam, The Netherlands Abbreviation. Pi/NaCI, phosphate-buffered saline (1 50 mM NaCl/10 mM potassium phosphate, pH 7.4). Enzyme. Glucocerebrosidase (EC 3.2.1.45).