Gaucher Disease (Glucocerebrosidase/13-Xylosidase/F-Glucosidase/Sphingolipidosis/Glucocerebroside) Yu-BIN CHIAO*, GREGORY M
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Proc. Nati. Acad. Sci. USA Vol. 75, No. 5, pp. 2448-2452, May 1978 Medical Sciences Multiple glycosidase deficiencies in a case of juvenile (type 3) Gaucher disease (glucocerebrosidase/13-xylosidase/f-glucosidase/sphingolipidosis/glucocerebroside) Yu-BIN CHIAO*, GREGORY M. HoYSON*, STEPHEN P. PETERS*, ROBERT E. LEE1, WARREN DIVENt, JEROME V. MURPHY1, AND ROBERT H. GLEW*§ Departments of *Biochemistry and t Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and * Department of Neurology, Milwaukee Children's Hospital, Milwaukee, Wisconsin 53233 Communicated by Albert L. Lehninger, January 16, 1978 ABSTRACT Biochemical investigations were performed nervous system involvement, Gaucher cells in nonneural tissues on autopsy tissues obtained from an 11-year-old girl who died (7), and survival after the 2nd year of life. with the juvenile, subacute neuropathic form of Gaucher dis- ease. In addition to the expected deficiency of glucocerebro- The biochemical basis that distinguishes the three clinical sidase activity, extracts of both liver and kidney from this in- forms of Gaucher disease is unknown. Brady and King (8) have dividual displayed a profound (>90%) deficiency of "soluble" suggested that the severity of the disease is related to the degree P-glucosidase, ft-xylosidase, and P-galactosidase activities. Fi- of deficiency of glucocerebrosidase activity. However, a broblasts obtained from this individual also contained markedly number of laboratories have not found a correlation between reduced levels of ,-xylosidase activity but normal levels of ft- the degree of deficiency of glucocerebrosidase activity and the D-fucosidase and P-galactosidase activity. Because the soluble -glucosidase,,-xylosidase, and a portion of the fl-galactosidase extent of pathology or organ involvement in various forms of activities from control human liver all cochromatographed on Gaucher disease (11-13). a gel filtration column of Sephadex G-200, it is suggested that Kanfer et al. (14) have put forth an alternate explanation to these activities all reside in a single enzyme, analogous to the account for the difference between infantile and adult Gaucher situation described in a number of nonhuman, mammalian disease. In addition to having the deficiency in particulate, tissues. ITis demonstration of multiple glycosidase deficiencies membrane-associated glucocerebrosidase, homogenates of brain in addition to the deficiency of glucocerebrosidase in a case of subacute neuropathic Gaucher disease suggests that other bio- and spleen but not liver from two infantile, neuropathic cases chemical aberrations, in addition to a deficiency of glucocere- of Gaucher disease were found to be profoundly deficient in brosidase, might contribute to pathology in some cases of a second, soluble (3-glucosidase (EC 3.2.1.21). The soluble fl- Gaucher disease. glucosidase, which is distinct from particulate glucocerebro- sidase, appears not to be deficient in the spleen of adults with Gaucher disease is an autosomal recessive inborn error of me- type I Gaucher disease (14, 15). The soluble f3-glucosidase tabojism characterized by the accumulation of glucocerebroside probably represents the aryl (3-glucosidase described by a in lysosomes of reticuloendothelial cells of affected individuals. number of investigators that will hydrolyze 4-methylumbelli- The glycosphingolipid accumulates as a result of a marked feryl-fl-D-glucopyranoside (16, 17) and steroid ,B-glucosides deficiency in glucocerebrosidase, the enzyme responsible for (14). its degradation (1). The diagnosis of Gaucher disease can be Ockerman has reported several cases of Gaucher disease of made from clinical signs and symptoms together with micro- unspecified clinical type in which the patients, in addition to scopic examination of a bone marrow aspirate for typical being deficient in particulate glucocerebrosidase, also appeared "Gaucher cells" (2), or by the determination of glucocere- to be deficient in the soluble aryl f-glucosidase (18, 19). An broside:,B-glucosidase (EC 3.2.1.45) activity using either a flu- extract of liver from one of these patients with Gaucher disease orogenic substrate (3-5) or radiolabeled glucocerebroside (6) that was profoundly deficient in soluble fl-glucosidase activity as substrate. was also markedly deficient in soluble fl-xylosidase activity (18); This sphingolipidosis presents most often in an adult, chronic, however, in this report the type of Gaucher disease that served nonneuropathic form (type 1) (7) characterized by spleno- as a source of enzyme was not indicated nor was specific activity megaly, hepatomegaly, anemia, thrombocytopenia, and erosion of (3-xylosidase compared in crude homogenates of control and of the cortices of the long bones (8). Patients with this form of Gaucher tissue. In the more recent study (19), homogenates of the disease display a wide variety of clinical severity: some in- liver from three children with an unspecified type of Gaucher dividuals are devastated by the disease as early as the 3rd dec- disease were shown to be profoundly deficient in 3-D-fucosidase ade of life while others have relatively symptom-free lives for activity; information concerning the f,-xylosidase content of 7 or 8 decades. Although a lack of neurologic involvement is these tissues was not provided. often cited as the distinguishing feature of the adult form of the Recently, Owada et al. (20) described four cases of acute disease, neurologic disorders in adults have been documented neuropathic and one case of subacute neuropathic Gaucher (9, 10). The second form of Gaucher disease is the rare, acute disease in which the activity of aryl fl-glucosidase was markedly neuropathic or infantile form (type 2) (7), which is character- deficient in extracts of spleen and fibroblasts. However, a de- ized by extensive neurologic involvement, hepato- ficiency in liver aryl fl-glucosidase was found in only two of splenomegaly, and death before age 2. Patients with juvenile these patients and both had the acute neuropathic form of or subacute neuropathic Gaucher disease (type 3) have central Gaucher disease. Glew et al. (17) have shown that a soluble aryl fl-glucosidase The costs of publication of this article were defrayed in part by the from rat kidney cortex has broad specificity in that the exten- payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. § To whom all correspondence should be addressed. 2448 Downloaded by guest on September 23, 2021 Medical Sciences: Chiao et al. Proc. Natl. Acad. Sci. USA 75 (1978) 2449 sively purified enzyme will hydrolyze fl-galactosides, (3-xy- buffer (0.3 M, pH 10.5) and liberated p-nitrophenol was esti- losides, and a-L-arabinosides, as well as f3-glucosides. It appears mated spectrophotometrically (E410, 14,300 M-1 cm'1). that this soluble aryl fl-glucosidase that is deficient in certain One unit of enzyme activity is defined as the amount of en- patients with acute or subacute neuropathic Gaucher disease zyme that cleaves 1 nmol of sugar from the appropriate sub- as described by Kanfer et al. (14), Ockerman and colleagues strate per hr at 37°. (18, 19), and Owada et al. (20) is analogous to the broad-spec- Protein Determination. Protein was estimated by the ificity aryl hydrolase described by Glew et al. (17). If this is true, method of Lowry et al. (23) with bovine serum albumin as a patients with a deficiency of soluble fl-glucosidase activity standard. should also be deficient in soluble (3-xylosidase and fl-galacto- Fibroblast Cultures. Fibroblasts were grown from skin ob- sidase activities. tained by biopsy or at autopsy in Eagle's minimum essential In the present report we describe the results of biochemical medium modified to contain 0.22% (wt/vol) NaHCO3, 1% investigations of extracts of liver, kidney, and fibroblasts from (wt/vol) nonessential amino acids, 10% (vol/vol) virus-screened a patient with subacute neuropathic Gaucher disease. These fetal calf serum, and antibiotics. Cells were grown at 370 in an tissues are shown not only to be glucocerebrosidase deficient, atmosphere of 95% air/5% CO2. As soon as growth of primary but also greater than 90% deficient in the activities of soluble cultures was well established, fibroblasts were subcultured into fl-glucosidase, fl-xylosidase, and f3-galactosidase. 250-ml Falcon flasks. When fully confluent, cells were removed by trypsinization, centrifuged at 250 X g for 10 min, and MATERIALS AND METHODS washed twice in phosphate-buffered saline, pH 7.4. Case Description. At birth, the patient was a 5-pound, 10-ounce (2.6 kg) female who was the product of an 8-month RESULTS pregnancy. She was noted to have a protuberant abdomen at 10 mo of age. At 2 yr of age strabismus on the left side and Multiple Glycosidase Deficiencies in Gaucher Liver and marked hepatosplenomegaly were evident. A bone marrow Kidney. As was described for leukocytes, glucocerebrosidase biopsy performed at 3 yr of age revealed the presence of activity was also markedly deficient in liver and kidney from Gaucher cells and splenectomy performed at that time yielded this patient with subacute neuropathic Gaucher disease. Her a 745-g spleen. Spasticity and opisthotonic posturing were noted liver contained 115 units of glucocerebrosidase activity per g at 33/4 yr and a seizure disorder began at age 4 yr. Immediately wet weight (control, 798 units/g); her kidney was similarly prior to her death at 11 yr of age, seizures were frequent, res- deficient in this activity. pirations were labored, and swallowing was poor. Table 1 shows the content of soluble hydrolases obtained from The glucocerebroside content of the patient's liver was 12.1 aqueous extracts of control and Gaucher liver and kidney. The mg/g wet weight, which is similar to the values reported by activities of (-glucosidase, O-xylosidase, and (3-galactosidase are others (7). Two years prior to her death, leukocyte glucocere- all markedly (290%) deficient in both Gaucher liver and kid- brosidase activity, determined according to the method of ney.