301

Biochimica et Biophysica Acta, 1096 (1991)301-311 © 1991 Elsevier Science Publishers B.V. 0925-4439/91/$03.50 ADONIS 092544399100082X

BBADIS 61044

Clinical phenotype of Gaucher disease in relation to properties of mutant glucocerebrosidase in cultured fibroblasts Sonja Van Weely 1, Marinella B. Van Leeuwen 1, Ineke D.C. Jansen 1, Marianne A.C. De Bruijn 1, Elisabeth M. Brouwer-Kelder 1, Andr6 W. Schram 1,., M. Clara Sa Miranda 2 John A. Barranger 3, Evelyn M. Petersen 4 Jack Goldblatt 4 Harald Stotz 5, Gi~nther Schwarzmann 5, Konrad Sandhoff 5, Lars Svennerholm 6, Anders Erikson 7, Joseph M. Tager 1 and Johannes M.F.G. Aerts 1 I E.C. Slater Institute for Biochemical Research, University of Amsterdam, Amsterdam (The Netherlands), 2 lnstituto Genetica MedicaJacinto Magalhaes, Porto (Portugal), 3 Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA (U.S.A.), 4 Department of Human Genetics, University of Cape Town (Republic of South Africa), 5 Institute for Organic Chemistry and Biochemistry, University of Bonn (F.R.G.), 6 Department of Neurochemistry, St. J6rgen's Hospital, Hisings Backa (Sweden) and 7 Barnkliniken, Centrallasarettet, Boden (Sweden)

(Received 9 November 1990) (Revised manuscript received 26 February 1991)

Key words: Glucocerebrosidase; Gaucher disease; Glucosylceramide lipidosis; Lysosomal hydrolase; Glycosphingolipidosis; Enzymic activity in situ

We have investigated several parameters of glucocerebrosidase in cultured skin fibroblasts from patients with various clinical phenotypes of Gaucher disease. In this study no strict correlation was found between the clinical manifestations of Gaucher disease and the parameters investigated in fibroblasts. These parameters included the specific activity of the enzyme in extracts towards natural lipid and artificial substrate in the presence of different activators; the enzymic activity per unit of glucocerebrosidase protein; the rate of synthesis of the enzyme and its stability; and the post-translational processing of the enzyme. In addition, the activity in situ of glucocerebrosidase in fibroblasts was investigated using a novel method by analysis of the catabolism of NBD-glucosylceramide in cells that were loaded with bovine serum albumin-lipid complexes. Again, no complete correlation with the clinical phenotype of patients was detectable. Glucocerebrosidase in fibroblasts from most non-neuronopathic (type 1) Gaucher disease patients differs in some aspects from enzyme in cells from patients with neurological forms (types 2 and 3). The stimulation by activator protein and phospholipid is clearly more pronounced in type 1 than in types 2 and 3; the enzymic activity per unit of glucocerebrosidase protein in type 1 is severely reduced in the presence of taurocholate and the amount of glucocerebrosidase appears (near) normal in contrast to the situation in types 2 and 3 Gaucher fibroblasts. However, this distinction was not always consistent; glucocerebrosidase in fibroblasts from some type 1 Gaucher patients, particularly some South African cases, was comparable in properties to enzyme in type 2 and 3 patients.

Introduction * Present address: Zaadunie, Enkhuizen, The Netherlands. Abbreviations: C6-NBD , 6-[N-7-nitrobenz-2-oxa-l,3-diazol-4-ylamino-caproyl]; Cer, ceramide; GD, Gaucher disease; GlcCer, glucosylceramide; fl-glu, glucocerebrosidase; fl-hex, fl-hexosaminidase; HMEM, Hepes buffered minimal essential medium; MU, methylumbelliferyl; 4-MU-fl-glu, 4-methylumbelliferyl-fl-D-glucoside; PAGE, polyacrylamide gel electrophoresis; PS, phosphatidylserine; SAP-2, sphingolipid activator protein 2 (also known as saposin C); SDS, sodium dodecyl sulphate; SM, sphingomyelin: Tch, taurocholate. Correspondence: J.M.F.G. Aerts, E.C. Slater Institute for Biochemical Research, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Gaucher disease, a recessively inherited lysosomal storage disorder, is highly variable in clinical expression [1]. On the basis of the extent and age of onset of primary neurological involvement three clinical phenotypes are generally distinguished: the non-neuronopathic variant (type 1 or adult form), the acute neuronopathic variant (type 2 or infantile form) and the subacute neuronopathic variant (type 3 or juvenile form). Type 1 Gaucher disease is the most prevalent form and the severity and clinical course of this variant is particu-

302 larly heterogeneous, ranging from early onset with severe splenomegaly and bone deterioration to no clinical manifestations [1]. Gaucher disease is characterised by lysosomal accumulation of glucosylceramide due to deficient activity of the hydrolase glucocerebrosidase (EC 3.2.1.45, D-glucosyl-N-acylsphingosine glucohydrolase) [2,3]. In recent years multiple defects in the structural gene encoding glucocerebrosidase have been identified in patients with Gaucher disease (Refs. 1, 4-8). Most of these mutations lead to a single amino acid substitution in the protein. One of the predominant mutations results in a substitution of Ser-370 for Asp-370 [5]. It has recently become clear that, although some Gaucher disease patients are homozygous for two identical mutant alleles, in most cases two distinct mutant alleles are present, i.e. the patients are genetic compounds with respect to glucocerebrosidase. This heterozygosity for any of the numerous 'Gaucher' mutations in combination with the observed hetero-allelism may partly explain the marked phenotypic variability of Gaucher disease [1]. No clear correlation has been shown between the molecular defect, enzymic properties or life history of glucocerebrosidase in Gaucher disease patients and the clinical expression of the disease [1]. In most patients with neurological involvement (types 2 and 3) there appears to be a reduction of glucocerebrosidase enzyme which correlates with the absence of an allele encoding for glucocerebrosidase mutated at position 370. In contrast, most patients with non-neuronopathic manifestations (type 1) have near normal to normal amounts of glucocerebrosidase which is associated with the presence of at least one allele encoding for enzyme mutated at position 370. However, it has previously been noted [9,10] that in some type 1 Gaucher disease patients a reduced amount of glucocerebrosidase protein is present. So far, attempts to correlate Gaucher disease phenotypes with properties of glucocerebrosidase have largely focused on the measurement of a single parameter of the enzyme, e.g., the nucleotide sequence of the alleles, the specific activity of the enzyme in vitro and the processing of the enzyme [1]. The activity in situ of glucocerebrosidase, i.e. the lysosomal activity in the intact cell, has received almost no attention due to the lack of a sensitive assay [11]. In this study we have concomitantly investigated multiple parameters of glucocerebrosidase in cultured skin fibroblasts from patients with various phenotypes of Gaucher disease. Moreover, the activity in situ of glucocerebrosidase in fibroblasts from a large group of patients has been assessed using a novel method. Materials and M e t h o d s

Materials. HAM FI0 culture medium, Hank's balanced salt solution, foetal calf serum, penicillin and

streptomycin were obtained from Flow (Irvine, Scotland); phosphatidylserine, leupeptin, 4-methylumbelliferyl substrates and bovine serum albumin from Sigma (St. Louis, U.S.A.); sodium taurocholate (grade A) from Calbiochem (San Diego, U.S.A.); Percoll, CNBractivated Sepharose 4B and protein A-Sepharose 4B from Pharmacia (Uppsala, Sweden); [35S]methionine from New England Nuclear (Boston, U.S.A.); Kieselgel 60 and Lichroprep RP-18 from Merck (Darmstadt, F.R.G.) and conduritol B-epoxide from Biomol Laboratories (Philadelphia, U.S.A.). All other reagents were of the purest grade available. A monospecific rabbit antiserum to placental glucocerebrosidase was prepared as described in Ref. 12. Monoclonal anti-(human glucocerebrosidase) antibodies 8E4 and 2C7 were obtained from mouse ascites [13]. Polyclonal anti-(cathepsin D) antibodies were obtained after immunisation of a rabbit with a pure preparation of the proteinase kindly provided by Dr. J.A. Barrett. Cell lines. In previous reports by our group a different nomenclature has been used for some of the fibroblast cell lines used in this investigation. Fibroblasts from normal individuals are referred to as: C-1 (previously Fc); C-2 (NK85AD); C-3 (LS); and C-4 (85AD5035). Fibroblasts from type 1 Gaucher disease patients are referred to as: lp-1 (185FF02); lp-2 (543LAD); lp-3 (TRIB86AD); lp-4 (MABO86AD); lp-5 (STECR86AD); lp-6 (CLYCL86AD); lp-7 (ISHEN86AD); lp-8 (ADSA86AD); lq-1 (PETAR86AD); lq-2 (WIBA86AD); lq-3 (ZBEL86AD); lq-4 (LEBO86AD) and lq-5 (PLONI86AD). A fibroblast cell line from a type 2 Gaucher disease patient is referred to as: 2-1 (GM877). Fibroblasts from type 3 Gaucher disease patients are referred to as: 3-1 (01674SF01); 3-2 (01297PF01); 3-3 (76RD26); and 3-4 (BAS85AD). Fibroblasts from Norrbottnian type 3 Gaucher disease patients are referred to as: 3-5 (F47) and 3-6 (627/83K). The phenotype of the Gaucher disease patients had been established by clinical examination. Methods Culture and extraction of cells. Cells were cultured in HAM F10 medium supplemented with 10% (by vol.) foetal calf serum, 14 mM bicarbonate, penicillin (250 I U / m l ) and streptomycin (250 ~tg/ml). Unless otherwise stated, the fibroblasts were harvested by trypsinisation 3-8 days after confluency. Cells were homogenised by sonication for 10 s in 50 mM potassium phosphate buffer (pH 6.0) containing 0.1% (by vol.) Triton X-100. The homogenate was centrifuged for 5 min at 10 000 × g and the supernatant was used. Enzyme assays. The enzymic activity of glucocerebrosidase in extracts from fibroblasts was measured under various conditions. Measurement of activity to-

303 wards glucosylceramide (14C-labelled in the glucose moiety) was performed as described in Ref. 14. The reaction mixture contained 60 mM potassium phosphate buffer (pH 5.8), 0.1% (by vol.) Triton X-100, 0.5% (mass/vol) sodium taurocholate and 400 /tM [14C]glucosylceramide. Measurement of glucocerebrosidase activity with 4-methylumbelliferyl-fl-glucoside as substrate in the presence of taurocholate was performed as described in Ref. 15. The reaction mixture contained 100/200 mM citrate/phosphate buffer (pH 5.2), 0.1% (by vol.) Triton X-100, 0.2% (mass/vol.) sodium taurocholate and 3.7 mM 4-methylumbelliferyl-fl-glucoside. Glucocerebrosidase activity towards 4-methylumbelliferyl-fl-glucoside was also measured in the presence of phosphatidylserine and sphingolipid activator protein preparation (SAP-2) as described in Ref. 16. The reaction mixture contained 50 mM acetate buffer (pH 5.0), 0.02% (by vol.) Triton X- 100, 2 0 0 / t g / # l phosphatidylserine and 0.1/~g/ml SAP-2. K m measurement. Glucocerebrosidase in extracts from fibroblasts was immunoprecipitated by incubation with anti-(human glucocerebrosidase) monoclonal antibodies 8E4 and 2C7 that had been immobilised to CNBractivated Sepharose 4B-beads. After extensive washing of the beads the enzymic activity of identical aliquots of bound enzyme was measured at different concentrations of 4-methylumbelliferyl-fl-glucoside (0.4-6 mM) in the presence of taurocholate.

Measurement of relative enzymic activity per amount of antigen (molecular activity). Two methods were used to determine the molecular activity of glucocerebrosidase. Method A has been described in detail in Ref. 17. Briefly, identical, limiting amounts of anti-(glucocerebrosidase) monoclonal antibody 8E4 are coated to wells of a microtitre plate. The wells are subsequently incubated with fibroblast extracts containing excess amounts of glucocerebrosidase protein. After washing and incubation with assay mixture the maximal bound enzymic activity per well is measured for each extract. Since the affinity of mutant and control glucocerebrosidase for binding to 8E4 is similar (as checked by mixing experiments) the activity that is bound to identical amounts of antibody is directly proportional to the enzymic activity per identical amount of antigen. In the second procedure (method B) wells of microtitre plates are coated with monoclonal antibody 8E4 and subsequently incubated overnight at 4°C and for 1 h at 37°C with a dilution range of fibroblast extracts. After washing of the wells the bound glucocerebrosidase activity is measured as in method A and subsequently the bound glucocerebrosidase molecules are quantified by incubation with a rabbit polyclonal anti-(glucocerebrosidase) antiserum, followed by incubation with donkey anti-(rabbit IgG) conjugated to Escherichia coli fl-galactosidase. The bound fl-galactosidase activity is measured by incubating the wells with a reaction mix-

ture containing 1.8 m g / m l 4-methylumbelliferyl-flgalactoside, 10 mM MgC12 and 100 m M fl-mercaptoethanol in 0.1 M sodium phosphate buffer (pH 7.3). No differences were observed when method B was performed with another anti-(glucocerebrosidase) monoclonal antibody (2C7) that recognises a different epitope on glucocerebrosidase [13]. Metabolic labelling of cultured skin fibroblasts. The procedure is described in Ref. 18. Fibroblasts were grown for 1 h in the presence of [35S]methionine. Next, the cells were harvested and extracted; glucocerebrosidase and cathepsin D were isolated by immunoprecipitation and subjected to polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS). The labelled proteins were quantified by scanning of fluorographs. Immunoblotting of glucocerebrosidase. Prior to polyacrylamide gel electrophoresis, fibroblast extracts were incubated overnight at 4°C with monoclonal antibodies 8E4 and 2C7 immobilised to CNBr-activated Sepharose 4B-beads. Subsequently, the beads were extensively washed in extraction buffer, resuspended in SDS-PAGE sample buffer and boiled for 5 min. After pelleting of the beads the supernatants were subjected to SDS-PAGE followed by immunoblotting as previously described [15]. Percoll gradient fractionation. Cells suspended in a solution containing 250 mM mannitol, 5 mM Mops and 1 mM E G T A were gently disrupted using a Dounce homogeniser and centrifuged for 2 min at 500 × g. The postnuclear supernatant obtained (1 ml) was applied on top of the Percoll layer (7 ml) adjusted to a density of 1.08 g/ml. After centrifugation for 1 h at 30 000 × g, fractions of 0.25 ml were collected. Enzymic activities of glucocerebrosidase, fl-hexosaminidase and other marker enzymes were measured in the presence of 0.1% (by vol.) Triton X-100 to disrupt compartments [19].

Measurements of in situ activity of glucocerebrosidase in intact cells. 6-[N-7-nitrobenz-2-oxa-l,3-diazol-4-ylaminocaproyl] sphingosyl-fl-glucoside (C6-NBD-glucosylceramide) was complexed to bovine serum albumin (fatty acid free) at a concentration of 5 /~M in Hepes buffered minimal essential medium (HMEM). Cultured skin fibroblasts were washed with H M E M and incubated with a glycolipid solution (10 nmol/25 cm 2 flask) for 1 h at 9°C. The cells were subsequently washed with H M E M and incubated further for 24 h at 37°C. About 90% of the total fluorescence in the pr~ paration was due to C6-NBD-glucosylceramide. Afte 24 h of incubation in the absence of cells the tota fluorescence was 90% of that seen initially and 90% of the total fluorescence was due to C6-NBD-glucosylceramide. The slight loss of the fluorescence during the chase period is due to instability of the NBD-substrate and not to formation of NBD-containing metabolites that are not recovered.

304 After harvesting the cells, glycolipids were extracted at 40°C in chloroform:methanol (1:1 by vol.). After dissolving in c h l o r o f o r m / m e t h a n o l / 0 . 1 M KC1 in H 2 0 (6 : 96 : 94 by vol.) it was applied to a Lichroprep RP-18 column. After washing with the same solvent and H20, lipids were eluted with methanol and c h l o r o f o r m / methanol (1 : 1 by vol.), successively. Glycolipids were separated by thin-layer chromatography using chlorof o r m / m e t h a n o l / 1 5 mM CaC12 in H 2 0 (60 : 35 : 8 by vol.) as developing system. NBD-containing lipids were visualised by ultraviolet illumination of the plates, the individual lipid species were extracted and fluorimetrically quantified. The fluorescence of the major spot seen at the front of the TLC plate was found to be not due to NBD; it was also consistently seen using extracts of cells that have never been exposed to NBD-substrates. The identity of the fluorescent compound is presently not known. The activity in situ of glucocerebrosidase is expressed as the ratio of fluorescence in ceramide and sphingomyelin to the total fluorescence (i.e. fluorescence in glucosylceramide, ceramide and sphingomyelin), since in this way variations due to different amounts of cells in the flasks harvested at different time points can be corrected for; C6-NBDceramide and C6-NBD-sphingomyelin are the main products formed (>_ 96%) after hydrolysis of C6-NBDglucosylceramide.

TABLE I

Specific activity of glucocerebrosidase and fl-hexosaminidase in fibroblast extracts The activity of glucocerebrosidase towards radioactively labelled glucosylceramide and 4-methylumbelliferyl-fl-glucoside in fibroblast extracts was measured in the presence of 0.5% and 0.2% (mass/vol.) taurocholate, respectively, as described in Materials and Methods. The activity of fl-hexosaminidase in extracts was measured using the corresponding 4-methylumbelliferyl substrate. All activities are expressed as n m o l / h per mg protein. The data were obtained from a single experiment in which all parameters were measured under identical experimental conditions. Exactly analogous results were obtained in other experiments in which some parameters were measured in individual cell lines or groups of cell lines. C, normal individuals; p and q, type 1 Gaucher patients. For more details concerning the type 1 p and q patients, see Table II and the text. Phenotype

Enzymic properties of glucocerebrosidase in fibroblast extracts The apparent K m value of glucocerebrosidase for 4-methylumbelliferyl-fl-glucoside in the presence of taurocholate and Triton X-100 was not significantly different for enzyme from patients with different phenotype or from control subjects (Table II). The ratio of enzymic activity towards 4-methylumbelliferyl substrate in the presence of phosphatidyl-

Substrate GlcCer

4MU-flghicoside

4-MU-fl-N-acetylglucosaminide

Control

C 1 2 3 4

575 281 291 296

589 313 356 282

7400 10400 9200 5000

Type 1

p 1 2 3 4 5 6 q 1 2 3 4 5

41 56 56 70 82 20 16 83 58 61 26

15 31 12 46 38 10 10 128 92 80 30

14100 8800 5300 14300 8200 3900 6200 14300 13200 9100 2600

Type 2

1

26

35

16300

Type 3

1 2 3 4 5 6

29 24 26 26 12 16

27 28 34 33 21 23

6200 10000 13700 8400 8600 8400

Results

Enzymic activity of glucocerebrosidase in extracts from cultured skin fibroblasts Table I shows that the specific activity of glucocerebrosidase in Triton X-100 extracts of fibroblasts from all Gaucher disease patients was reduced, whereas that of another lysosomal hydrolase (/3-hexosaminidase) was normal. It can be seen in the case of some type 1 patients (defined below as lp) that the activity towards the natural substrate was higher than towards the artificial, 4-methylumbelliferyl substrate. This is due to the different taurocholate concentrations in the assays. Enzyme from type lp patients requires a relatively high concentration of taurocholate for maximal activity (results not shown). Table I shows that no clear correlation exists between the clinical phenotype of Gaucher disease and residual specific activity of glucocerebrosidase.

No.

serine and sphingolipid activator protein (SAP) related to the enzymic activity in the presence of taurocholate was very similar to control values in the case of glucocerebrosidase in extracts from t y p e 2 and 3 Gaucher disease patients (Table II). However, in the case of some type 1 Gaucher disease patients, referred to as type lp, the enzyme is clearly more active in the presence of phosphatidylserine and SAP than in the presence of taurocholate. Very similar findings have been made for the mutant enzyme in urine samples of all investigated Dutch type 1 Gaucher disease patients (n = 20), for enzyme in leukocytes from all investigated Portuguese type 1 Gaucher disease patients (n = 8) and for enzyme from spleens of Caucasian and Jewish type 1 Gaucher disease patients (n = 5). A similar effect has previously been reported by other investigators [20]. In

305 TABLE II

Enzymic properties of glucocerebrosidase in fibroblast extracts The ethnic background of subjects is described as follows: European caucasian (e); African black (ab); African white (aw); Cape coloured (cc); American caucasian (a). K m values of glucocerebrosidase for its 4-methylumbelliferyl substrate were measured in the presence of 0.2% (mass/vol.) taurocholate after immunopurification of the enzyme from fibroblast extracts as described in Materials and Methods. The activity of glucocerebrosidase in the presence of phosphatidylserine (PS) and activator protein preparation (SAP) and in the presence of taurocholate (Tch) was measured as described in Materials and Methods. Actual values for activities with 4 MU/Tch are given in Table I. The values for activities with 4 MU/PS-SAP can be calculated. The K m values and activities with 4 MU/PS-SAP and 4 MU/Tch were results of a typical experiment. Similar data were obtained in other independent experiments using individual cell lines or groups of cell lines. The molecular activity of glucocerebrosidase in fibroblast extracts was related to that of enzyme in extracts from fibroblasts of a normal individual (C-l). Molecular activity of glucocerebrosidase was determined via two methods (A and B), exactly as described in Materials and Methods. Phenotype

No.

Ethnic background

Km 4MU-flglu (mM)

(4MU/PS-SAP)/ (4MU/TCH)

Molecular activity (%) Method A

Method B

mean+ S.E.

(n)

mean + S.E.

(n)

Control

C1 2 3

e e ab

2.8 2.1

0.72 0.76 0.84

100 108+ 3 91

(11) (3) (1)

100 120 78 + 26

(9) (1) (3)

Type 1

p1 2 3 4 5 6 7 q1 2 3 4 5

e e e aw aw cc cc cc cc ab ab ab

3.2 3.2 2.6 2.6 2.5 3.7

3.13 2.79 5.00 1.94 3.15 4.00

3.3 2.2 2.4 2.4 2.4

1.01 0.67 0.74 0.86 0.83

11 + 2 9+0 5+0 13 + 2 9+0 5 +0 5+ 0 41 + 1 69 + 2 62 + 5 61 + 4 48 + 7

(9) (3) (3) (4) (3) (3) (3) (3) (3) (4) (4) (3)

14+ 3 7, 13 4, 10 19 + 5 9, 14 5, 14 5, 9 58 69, 80 122 + 5 115, 119 85

(7) (2) (2) (3) (2) (2) (2) (1) (2) (3) (2) (1)

Type 2

1

a

2.3

0.72

25, 56

(2)

48 _ 3

(3)

Type 3

1 2 3 4 5 6

e e e e e e

2.2 2.4 2.6 2.2

0.77 0.86 0.86 0.85 0.65 0.69

64 + 3 31, 71 68+2 39, 57 46 52

(6) (2) (3) (2) (1) (1)

55 + 10 65 82, 113 77 46, 89 52, 115

(4) (1) (2) (1) (2) (2)

contrast, enzyme from some South African type 1 G a u c h e r d i s e a s e p a t i e n t s ( r e f e r r e d to as t y p e l q ) was not more stimulated by phosphatidylserine and activator p r o t e i n t h a n b y t a u r o c h o l a t e . A l l p a t i e n t s t h a t w e r e f o u n d to b e l o n g to t y p e l q in this s t u d y are o f (partial) black ancestry. The enzymic activity of glucocerebrosidase per unit o f c r o s s - r e a c t i v e m a t e r i a l ( r e f e r r e d to as t h e m o l e c u l a r activity) was determined by two different immunol o g i c a l p r o c e d u r e s as d e s c r i b e d in M a t e r i a l s a n d M e t h ods. T a b l e II s h o w s t h a t t h e m o l e c u l a r a c t i v i t y o f g l u c o c e r e b r o s i d a s e as m e a s u r e d in the p r e s e n c e o f t a u r o c h o l a t e was m a r k e d l y r e d u c e d o n l y in the c a s e o f t y p e lp Gaucher disease patients.

Synthesis and processing of glucocerebrosidase in fibroblasts The synthesis and processing of glucocerebrosidase in f i b r o b l a s t s f r o m c o n t r o l s u b j e c t s a n d f r o m s o m e

patients with different phenotypes of Gaucher disease h a v e b e e n s t u d i e d e x t e n s i v e l y b y us a n d b y o t h e r s ( f o r a r e v i e w see R e f s . 1, 18, 21 a n d 22). G h i c o c e r e b r o s i d a s e does not undergo proteolytic modification, with the e x c e p t i o n o f the r e m o v a l o f t h e h y d r o p h o b i c l e a d e r , b u t is s u b j e c t to e x t e n s i v e m o d i f i c a t i o n o f its o l i g o s a c c h a ride chains. T h e initial f o r m o f g l u c o c e r e b r o s i d a s e o b served during metabolic labelling experiments has an apparent M r of 62500 and contains four highm a n n o s e - t y p e o l i g o s a c c h a r i d e chains. A f t e r 1 - 3 h this f o r m is g r a d u a l l y c o n v e r t e d to a d i f f u s e 66 k D a f o r m that represents enzyme with variably sialylated, comp l e x - t y p e o l i g o s a c c h a r i d e chains. T h i s f o r m is e v e n t u ally c o n v e r t e d to a 59 k D a f o r m b y l y s o s o m a l e x o g l y cosidases. It has p r e v i o u s l y b e e n n o t e d t h a t t h e a m o u n t o f g l u c o c e r e b r o s i d a s e p r o t e i n s y n t h e s i s e d in f i b r o b l a s t s f r o m p a t i e n t s w i t h v a r i o u s p h e n o t y p e s o f G a u c h e r dise a s e is s i m i l a r to t h a t in cells f r o m c o n t r o l s u b j e c t s [1,18,21]. S i m i l a r results w e r e o b t a i n e d w i t h a d d i t i o n a l

306 T A B L E III

Biosynthesis of glucocerebrosidase as related to that of cathepsin D in fibroblasts The biosynthesis of glucocerebrosidase (/3-glu) and cathepsin D was measured by metabolic labelling of proteins with [35S]methionine for 1 h, followed by immuno-isolation of the two proteins, as described in Materials and Methods. The ratio of synthesis of glucocerebrosidase to that of cathepsin D was determined by scanning of fluorographs of purified glucocerebrosidase and cathepsin D that had been subjected to SDS-PAGE. The data represent values obtained from scanning 4 differently exposed fluorographs of one experiment. Similar results were obtained in a second experiment. Phenotype

No.

fl-glu signal/cathepsin D signal ( × 1000) mean _+S.E.

Control

C 1 2

73 _+21 83+17

Type 1

p 1 2 4 q 1 2 3 4

157+51 257+57 86+24 157+35 127+32 150+37 117_+35

Type 2

1

89 + 18

Type 3

1 2 3 4

84 5:19 6 0 + 19 48+14 53+19

cell lines in this study. As shown in Table III, the ratio radioactivity in glucocerebrosidase/radioactivity in cathepsin D after labelling cells for 1 h with [35S]methionine was similar in control and mutant cell lines. In previous studies emphasis has been placed on the differences in the amount of cross-reactive material and the pattern of molecular mass forms of glucocerebrosidase in relation to the clinical phenotype of Gaucher disease (for a review see Refs. 1 and 18). We have now extended these studies to include a larger number of patients and have observed that the type l q patients are

characterised by a marked decrease in glucocerebrosidase protein similar to that seen in the neurological phenotypes (see Fig. 1A). In addition, by analysing immunoprecipitates instead of total extracts we have been able to visualise the pattern of molecular mass forms of glucocerebrosidase in cells containing little cross-reactive material more accurately than previously [12,18]. In all cell lines studied the 59 kDa form of glucocerebrosidase was detectable (see Fig. 1 B).

Subcellular localisation of glucocerebrosidase in fibroblasts as revealed by Percoll density gradient fractionation In a previous immunocytochemical study it was found that the amount of glucocerebrosidase in lysosomes of cultured skin fibroblasts is markedly reduced in the case of type 2 and 3 Gaucher disease patients, but not in the case of type 1 Gaucher patients [23]. We have therefore investigated the subcellular localisation of glucocerebrosidase using Percoll density gradient fractionation of cell homogenates. Table IV shows the distribution of glucocerebrosidase activity between the pooled light fraction, which contains prelysosomal compartments and probably newly formed lysosomes, and the heavy fraction, which contains mature lysosomes. The glucocerebrosidase in fibroblasts from control subjects and patients with various phenotypes of Gaucher disease shows a very similar distribution to that of fl-hexosaminidase over the two fractions. This implies that the small amount of residual glucocerebrosidase in cells from type lq, type 2 and type 3 Gaucher disease patients is transported to lysosomes and must possess a near-normal stability in mature lysosomes. This is also suggested by the presence of the lysosomal 59 kDa form of the enzyme in cells from these patients; this form arises by the action of exoglycosidases in lysosomes [221. The effects of sucrose and low temperature on glucocerebrosidase levels in fibroblasts The intracellular level of glucocerebrosidase can be manipulated by the conditions under which cells grow. We have noted that glucocerebrosidase is stabilised in 1

A

1

2

3

4

5

6

7

8

2

3

4

5

9

68-68

--

....... ~

; ~!~: o

59

•

.....

59--

•

1

Fig. 1. (A) Pattern of molecular mass species of glucocerebrosidase in fibroblasts from some control subjects and patients with different Gaucher disease phenotypes. Prior to SDS-PAGE and immunoblotting, glucocerebrosidase was purified by immunoprecipitation from an aliquot of fibroblast extract containing 0.2 m g total protein. Lane 1, control, no. C-l; lane 2, control, no. C-3; lane 3, type l p G D , no. 1; lane 4, type l p G D , no. 4; lane 5, type l q G D , no. 3; lane 6, type l q GD, no. 4; lane 7, type 2 G D , no. 1; lane 8, type 3 GD, no. 1; lane 9, type 3 G D , no. 5. Abbreviation: G D , Gaucher disease. (B) Molecular mass species of glucocerebrosidase in fibroblasts from some patients with reduced a m o u n t s of antigen. Prior to SDS-PAGE and immunoblotting, glucocerebrosidase was purified by immuno-precipitation from an extract containing 1 m g of protein. Lane 1, type l q G D , no. 3; lane 2, type 2 G D , no. 1; lane 3, type 3 G D , no. 1; lane 4, type 3 G D , no. 5; lane 5, type 3 GD, no. 6.

307 T A B L E IV

TABLE V

Distribution of glucocerebrosidase and fl-hexosaminidase activity upon Percoll density gradient fractionation

Effect of hyperosmolar culture conditions on glucocerebrosidase level in fibroblasts

Percoll gradient fractionation of subcellular compartments in homogenates of fibroblasts was performed as described in Materials and Methods. In all experiments, the activity of ghicocerebrosidase (fl-glu) and fl-hexosaminidase (fl-hex) recovered in the cytosolic fraction due to disrupted compartments was less than 15%. The activities of both hydrolases showed the dual distribution typical for lysosomal enzymes on Percoll gradients. LL, represents the activity of hydrolases in fractions with densities ranging from 1.02 to 1.05 g / m l ; HL, represents the activity of hydrolases in fractions with densities ranging from 1.06 to 1.17 g / m l . The values represent the means- S.E. of the indicated number of independent experiments.

Ceils were grown for 14 days in iso-osmolar m e d i u m or medium made hyperosmolar by the additional presence of 80 m M sucrose. The activity of glucocerebrosidase in cells was determined in extracts using 4-methylumbelliferyl-fl-glucoside as substrate in the presence of 0.2% (mass/vol.) taurocholate. The values represent the cellular glucocerebrosidase level in fibroblasts grown under hyperosmolar conditions compared to that in cells grown under iso-osmolar conditions. The results of a typical experiment are indicated. Similar data were obtained in other independent experiments using these and additional cell lines.

Phenotype

No.

Relative distribution Relative distribution of fl-glu in gradient of fl-hex in gradient LL

HL

LL

HL

Control

C 1 2 3 5

23+2 23+3 32 16

77+1 77+3 68 84

16+2 9+1 26 13

84+1 91+1 74 87

12 3 1 1

Type 1

p 1 2 4 q 1 2 3

23+3 23 40 20, 29 28 39

77+3 77 60 80, 71 72 61

15+2 15 23 12, 9 12 37

85+2 85 77 88, 91 88 63

5 1 1 2 1 1

Type2

1

41+11

59+11

175-1

835-1

3

Type 3

1 2 3 4 5

24+4 285-8 205-0 11, 18 48

76+4 725-8 805-0 89, 82 52

185-1 11+1 95-1 3, 5 19

825-1 895-1 915-1 97, 95 81

3 6 4 2 1

Phenotype

No.

Relative increase of activity by hyperosmolarity

Control

C 1

1.9

Type 1

p 1 4 q 1 3 4

1.7 1.9 6.3 2.6 3.1

Type 2

1

3.8

Type 3

1 2 3

4.5 2.6 3.6

n

cells grown under hyperosmolar conditions. This is probably due to a marked decrease in transport of newly formed enzyme from the trans Golgi region to mature lysosomes (J.M.F.G. Aerts and S. Van Weely, unpublished data). When fibroblasts were grown in medium made hyperosmolar by the additional presence of 80 mM sucrose, the cellular glucocerebrosidase levels were increased. Table V shows that the increase in glucocerebrosidase was similar in fibroblasts from control subjects and type l p Gaucher disease patients. However, hyperosmolarity led to a more marked increase in glucocerebrosidase levels in fibroblasts from most type lq, type 2 and type 3 Gaucher disease patients investigated. This suggests that in the latter cells under hyperosmolar conditions more glucocerebrosidase protein was present due to inhibition of degradation. We also observed that culturing of cells at 19°C resulted in stabilisation of glucocerebrosidase in some of the cell lines from type 2 and 3 Gaucher disease patients. In these experiments, fibroblasts were first pre-incubated with conduritol B-epoxide, an irreversible

inhibitor of glucocerebrosidase, to inactivate all the enzyme present. Subsequently, the cells were grown at 19°C for 11 days and the glucocerebrosidase activity was measured in extracts of the cells. Table VI shows that under these conditions the glucocerebrosidase level in cells from the type 3 Gaucher disease patient was very similar to that of a control subject. The glucocerebrosidase level in the investigated cells from the type 2 Gaucher disease patient was also relatively high compared to that in the normal culture situation (see Table I). However, in the case of cells from the type l p Gaucher disease patient the glucocerebrosidase level remained deficient to the same extent as under isoosmolar culture conditions (cf. Tables I and VI). An TABLE VI

Effect of culturing at 19°C on glucocerebrosidase level in fibroblasts Prior to the experiment the glucocerebrosidase in cells was inactivated by incubation with 0.1 m M conduritol B-epoxide. After removal of excess inhibitor by extensive washing cells were grown at 19°C for 11 days. The cells were harvested and the glucocerebrosidase activity was determined in extracts as described in the legend to Table V. Phenotype

No.

Glucocerebrosidase activity ( n m o l / h per mg)

Control Type 1 Type 2 Type 3

C 1 p 1 1 1

9.1 0.7 1.8 6.9

308 explanation for these findings is suggested by the observation that in cells grown at 19°C the initial (62.5 kDa) form of glucocerebrosidase accumulated in compartments with low density, suggesting a block in transport of the enzyme to the trans Golgi region. Thus, inhibition of cellular transport processes at a temperature of 19°C seemed to prevent rapid degradation of glucocerebrosidase in the cells from the type 2 patient and, in particular, the type 3 Gaucher disease patient studied. The glucocerebrosidase activity was not increased in the case of cells from the type l p Gaucher disease patient, since the deficient activity in this case is not due to an instability of the enzyme, but to a reduced enzymic activity per amount of glucocerebrosidase protein (Table II).

ST

1

2

3

4

5

6

--

GM3

The activity in situ of glucocerebrosidase towards exogenous NBD-labelled glucosylceramide The enzymic activity of glucocerebrosidase towards glucosylceramide in lysosomes of intact fibroblasts was measured using a novel method that is described in Materials and Methods. Fibroblasts were exposed for 1 h at 9°C to C6-NBD-glucosylceramide complexed to bovine serum albumin, allowing insertion of the lipid in the plasma membrane. Subsequently, after removal of remaining exogenous lipid, the fibroblasts were grown for 24 h at 37°C to allow delivery of the glycolipid to lysosomes and intralysosomal degradation. The cells were harvested and the NBD-containing lipids were quantified after extraction and thin-layer chromatography. Fig. 2 shows that glucosylceramide was mainly catabolised to ceramide which was subsequently converted to sphingomyelin. Under the experimental conditions used, metabolism of glucosylceramide must almost completely have been dependent on the action of lysosomal glucocerebrosidase, since it was found to be nearly completely inhibited by conduritol B-epoxide. Conduritol B-epoxide is a specific inhibitor of lysosomal glucocerebrosidase and has no effect on other ]3-glucosidases, at least in mammalian cells [24]. Using radioactively labelled natural glucosylceramide instead of its NBD-analogue we observed that the lipid was mainly anabolised by the cells and that only little of the internalised glucosylceramide was hydrolysed by glucocerebrosidase. Apparently, only a small proportion of this labelled glucosylceramide is delivered to lysosomes via the approach used. A similar problem has previously been encountered by other investigators [11]. Table VII shows that no clear relationship was found between the clinical phenotype of Gaucher disease and the activity in situ in cultured skin fibroblasts.

--

GM2

Discussion

ST

-- Cer

--

GlcCel

- - LacCe~

j SM

-- GM1

--

GD1 a

--ORIGIN

Fig. 2. Metabolism of C6-NBD glucosylceramide in fibroblasts. Comparable amounts of fibroblasts from different individuals were loaded with C6-NBD-glucosylceramide for 1 h at 9°C and subsequently incubated for 24 h at 37°C without further addition of glycolipid, exactly as described in Materials and Methods. Cells were harvested and glycolipids were extracted and subjected to thin layer chromatography exactly as described in Materials and Methods. NBD-containing lipids on the thin layer plate were visualised by ultraviolet illumination. Cer, ceramide; GlcCer, glucosylceramide; LacCer, lactosylceramide; and SM, sphingomyelin. Lane 1, control, no. C-l; lane 2: type lp GD, no. 1; lane 3, type l q GD, no. 2; lane 4, type l q GD, no. 3; lane 5, type 2 GD, no. 1; lane 6, type 3 GD, no. 1; ST: glycolipid standards. The arrowhead indicates a fluorescent spot unrelated to N B D and consistently seen in fibroblast extracts.

None of the parameters of glucocerebrosidase that were examined in this study fully discriminated between the relatively mild non-neuronopathic type 1 variant of Gaucher disease and the severe types 2 and 3. This investigation confirmed our previous studies showing that glucocerebrosidase from the majority of type 1 Gaucher disease patients (n = 33), referred to as type lp, had distinct properties: the relative enzymic activity per amount of antigen measured in the presence of taurocholate was severely reduced and the enzymic activity was greatly stimulated by phospholipid in combination with activator protein SAP-2. Concomitantly, the biosynthesis, processing, intracellular transport and stability of the enzyme in fibroblasts appeared (near) normal. In this study, the properties of glucocerebrosidase from five of the type 1 Gaucher disease cases proved to be clearly different in that the relative enzymic activity per amount of antigen was not severely reduced, the

309 T A B L E VII Residual activity of glucocerebrosidase in Gaucher disease fibroblasts as measured in situ and in extracts of cells The activity in situ of glucocerebrosidase towards NBD-labelled glucosylceramide was determined exactly as described in Materials and Methods. The activity of glucocerebrosidase in extracts of cells was determined using 4-methylumbelliferyl-fl-glucoside as substrate in the presence of taurocholate as described in Materials and Methods. The values of activities in Gaucher disease fibroblasts are related to those measured in cells from a normal individual (C-1). Activity was determined as the ratio of fluorescence in ceramide and sphingomyelin to the total fluorescence (i.e. fluorescence in glucosylceramide, ceramide and sphingomyelin; fluorescence in other lipids was always less than 4%). Phenotype

No.

Residual activity (% of control C-l) in situ with NBD-GIcCer (mean_+ S.E. (n))

in extracts with 4MU-flGlu/Tch (mean + S.E. (n)) 100 145+38 89_+ 10

Control

C 1 C 2 C 3

100 105 75

Type 1

p 1 p 8 q 1 q2 q3

44_+ 3 28_+ 4 18 72, 88 109_+ 8

Type2 Type3

1

(14) (1) (1) (3) (3) (1) (2) (3)

27_+ 6 (7)

6_+ 6 6_+ 30_+ 38_+

1

(21) (3) (4)

1 2 7

(15) (1) (6) (4) (7)

6_+ 1

(11) (14)

3

57_+12 (4)

14_+ 2

5

35

(1)

11_+ 3

(3)

6

54

(1)

12+ 4

(3)

enzyme was not strongly stimulated by phospholipid and activator protein and the stability of the enzyme appeared to be low. Indeed, the properties of glucocerebrosidase in this group of patients (designated as type lq) could not be clearly distinguished from those of enzyme from patients with neuronopathic Gaucher disease. On the basis of our present data it seems that fibroblasts with (near) normal amounts of glucocerebrosidase protein showing the distinct kinetic properties described above indicate for a non-neuronopathic form of Gaucher. However, the number of neurologically affected cases studied so far is too small to consider this a safe diagnostic criterion. In contrast, as clearly shown in this study and previously [9,10] reduced amounts of glucocerebrosidase protein in cultured skin fibroblasts does not warrant an unequivocal prediction of the development of neurological complications during the course of Gaucher disease. Grabowski et al. [25,26] classified type 1 Gaucher disease patients into two groups on the basis of some properties of glucocerebrosidase [25,26]. In their classification, enzyme from type 1 A Gaucher disease patients is similar in several kinetic properties to the enzyme from control subjects and that from type 2 and type 3

Gaucher disease patients; enzyme from (almost exclusively) Ashkenazi Jewish type 1 Gaucher disease patients (referred to as type 1B) was found to show characteristically distinct kinetic properties. The enzyme from the latter group was relatively more stimulated by taurocholate or phosphatidylserine and was less sensitive to inhibition by conduritol B-epoxide or glucosylsphingosine. Enzyme from patients classified by us as types lp and lq, seems analogous to enzyme from patients classified by Grabowski et al. as type 1B and type 1A, respectively. Indeed, enzyme from type l p patients had different responses to conduritol B-epoxide and glucosylsphingosine from that of other patients (not shown), analogous to the findings made by Grabowski for enzyme from type 1B patients. However, it is remarkable that in our experience all type 1 Gaucher disease patients in The Netherlands and Portugal (n = 28) and the small number of studied cases from France and the U.S.A. (n = 8) belong to the type lp category. As the majority of type l p patients are not of Ashkenazi Jewish ancestry, this form of glucocerebrosidase is not specific to Ashkenazi Jewish type 1 Gaucher disease patients. This observation is substantiated by recent findings on the nature of mutations in glucocerebrosidase alleles in type 1 Gaucher disease patients from different ethnic backgrounds. It was found that the same mutations in glucocerebrosidase alleles occur in Ashkenazi and non-Ashkenazi individuals and that the mutation resulting in substitution of Ser-370 for Asp is particularly frequent, being present, as a single copy at least, in most type 1 Gaucher disease patients [5]. Most likely the prevalence of this particular mutation underlies the finding that glucocerebrosidase from most type l(p) Gaucher disease patients has similar abnormal kinetic properties. Mouse 3T3 fibroblasts have been transfected with cDNA encoding for glucocerebrosidase with this particular mutation. Subsequent purification and analysis of the human enzyme expressed in the mouse cells showed that this mutant protein is stable, but that the activity per molecule of enzyme protein is very low. Most other mutations in glucocerebrosidase alleles that have been identified so far lead to the formation of glucocerebrosidase that is rapidly degraded [27]. Consequently, in most type 1 Gaucher disease patients the residual glucocerebrosidase protein will consist almost entirely of the 370-mutated enzyme molecules. This explains the observed similarity of residual glucocerebrosidase from most type 1 Gaucher disease patients. Since the five type l q patients did not have stable glucocerebrosidase protein with abnormal kinetic properties, it appears most likely that these individuals do not carry an allele encoding for 370-mutated glucocerebrosidase or any other mutation leading to a similar type of stable mutant enzyme. Analysis of the nucleotide sequence of glucocerebrosidase alleles from these

310 patients is in progress. It must be noted that all type l q patients in this study were individuals of (partial) black ancestry. Very recently it has been reported that a large proportion of Japanese type 1 Gaucher disease patients studied are homo-allelic for the glucocerebrosidase allele encoding for enzyme mutated at position 444 [28]. This is remarkable, since in other studies concerning patients from Caucasian and Jewish ancestry it has so far always been found that individuals homo-allelic for this mutant glucocerebrosidase allele develop severe neurological symptoms [4,8]. Of interest in this respect is the recent finding that the patients with the Norrbottnian form of Gaucher disease are homo-allelic for the Leu-444 ---, Pro-444 mutation [29]. Thus, it appears that epistatic mechanisms in some instances strongly influence the clinical manifestations of Gaucher disease amongst patients with identical glucocerebrosidase alleles. The novel method described in this report, using NBD-labelled glucosylceramide as substrate, allows measurement of enzymic activity of glucocerebrosidase in lysosomes of intact fibroblasts. The residual enzymic activity of glucocerebrosidase measured in this way in cells from severely and mildly affected patients was not found to be different. A number of explanations are possible for this finding. Firstly, it is conceivable that the catabolism of the lipid analogue differs essentially from that of the natural lipid substrate or that the loading procedure employed delivers the lipid in an abnormal state to lysosomes. Secondly, fibroblasts may be an unattractive cell model for studies concerning Gaucher disease. The level of glucocerebrosidase in cultured skin fibroblasts is about 30-fold higher than that in blood cells and most tissues. This implies that, even in fibroblasts from type 2 and 3 Gaucher disease patients that are severely deficient in glucocerebrosidase protein, more enzyme is present per unit of total cellular protein than in normal blood cells and tissues. Finally, we have noted that glucocerebrosidase from the majority of type 1 Gaucher disease patients is nearly normal in enzymic activity in the presence of appropriate amounts of activator protein and phospholipid. Aggregates of glucocerebrosidase and activator protein have been detected in extracts from cells and tissues. Only small amounts of such aggregates are found in extracts of fibroblasts but extracts of blood cells or transformed lymphocytes are relatively rich in this form of glucocerebrosidase. It is therefore not surprising that the residual activity of glucocerebrosidase in the absence of additional detergents or activators is relatively high in extracts from leukocytes from type 1 Gaucher disease patients, but not in those from fibroblasts from the same patients [30]. In preliminary experiments we have noted that the measurement of glucocerebrosidase activity in situ using NBD-labelled glucosylceramide gives different results when transformed lymphocytes are

compared to fibroblasts. The residual activity of glucocerebrosidase in situ does, indeed, appear to be relatively high in transformed lymphocytes from type l(p) Gaucher disease patients in contrast to the situation in fibroblasts from the same patients. In conclusion, the findings made in this investigation as well as those in other recent studies concerning glucocerebrosidase alleles in patients with Gaucher disease imply that additional factors besides glucocerebrosidase contribute to the clinical manifestation of Gaucher disease. Knowledge about the ganglioside catabolism in situ in cell types that are more relevant in Gaucher disease than fibroblasts is needed for a better insight into the clinical expression of Gaucher disease.

Acknowledgements We are most grateful to Mr. Niels Galjart, Ms. Anneke Strijland and Dr. Lisbeth Jonsson for performing preliminary experiments. We would like to thank Ms. Wendy van Noppen and Ms. Elsbeth van Vlugt-van Daalen for their help in the preparation of this manuscript. This study was supported by a generous grant from the National Gaucher Foundation, U.S.A. (grant N G F 17). J.M.F.G. Aerts is an established investigator of the Royal Netherlands Academy of Sciences.

References 1 Barranger, J.A. and Ginns, E.I. (1989) in The Metabolic Basis of Inherited Disease, (Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D., eds), pp. 1677-1698, Mc Graw-Hill, New York. 2 Brady, R.O., Kanfer, J.N. and Shapiro, D. (1965) Biochem. Biophys. Res. Commun. 18, 221-225. 3 Patrick, A.D. (1965) Biochem. J. 97, 17c-17d. 4 Tsuji, S., Choudary, P.V., Martin, B.M., Stubblefield, B.K., Mayor, J.A., Barranger, J.A. and Ginns, E.I. (1987) N. Engl. J. Med. 316, 570-575. 5 Tsuji, S., Martin, B.M., Barranger, J.A., Stubblefield, B.K., LaMarca, M.E. and Ginns, E.I. (1988) Proc. Natl. Acad. Sci. USA 85, 2349-2352. 6 Zimran, A., Gross, W., West, C., Sorge J., Kubitz, M. and Beutler, E. (1989) Lancet 8659, 349-352. 7 Theophilus, B.D.M., Latham, T., Grabowski, G.A. and Smith, F.I. (1989) Am. J. Hum. Genet. 44, 349-352. 8 Firon, N., Eyal, N., Kolodny, E.H. and Horowitz, M. (1990) Am. J. Hum. Genet. 46, 527-532. 9 Fabbro, D., Desnick, R.J. and Grabowski, G.A. (1987) Am. J. Hum. Genet. 40, 15-31. 10 Van Weely, S., Aerts, J.M.F.G., Van Leeuwen, M.B., Peterson, M.E., Goldblatt, J., Tager, J.M., Barranger, J.A. and Schram, A.W. (1987) in Lipid Storage Disorders (Salvayre, R., Douste-Blazy, L. and Gatt, S., eds.), pp. 51-56, Plenum Press, New York. 11 Saito, M. and Rosenberg, A. (1985) J. Biol. Chem. 260, 2295-2300. 12 Ginns, E.I., Brady, R.O., Pirruccello, S., Moore, C., Sorrell, S., Furbish, F.S., Murray, G.J., Tager, J. and Barranger, J.A. (1982) Proc. Natl. Acad. Sci. USA 79, 5607-5610. 13 Barneveld, R.A., Tegelaers, F.P.W., Ginns, E.I., Visser, P., Laanen, E.A., Brady, R.O., Galjaard, H., Barranger, J.A., Reuser, A.J.J. and Tager, J.M. (1983) Eur. J. Biochem. 134, 585-589.

311 14 Furbish, F.S., Blair, H.E., Shiloach, J., Pentchev, P.G. and Brady, R.O. (1977) Proc. Natl. Acad. Sci. USA. 74, 3560-3563, 15 Aerts, J.M.F.G., Donker-Koopman, W.E., Van der Vliet, M.K., Jonsson, L.M.V., Ginns, E.I., Murray, G.J., Barranger, J.A., Tager, J.M. and Schram, A.W. (1985) Eur. J. Biochem. 150, 565-574. 16 Aerts, J.M.F.G., Sa Miranda, M.C., Brouwer-Kelder, E.M., Van Weely, S., Barranger, J.A. and Tager, J.M. (1990) Biochim. Biophys. Acta 1041, 55-63. 17 Aerts, J.M.F.G., Donker-Koopman, W.E., Brul, S., Van Weely, S., Sa Miranda, M.C., Barranger, J.A., Tager, J.M. and Schram, A.W. (1990) Biochem. J. 269, 93-100. 18 Jonsson, L.M.V., Murray, G.J., Sorrell, S.H., Strijland, A., Aerts, J.M.F.G., Ginns, E.I., Barranger, J.A., Tager, J.M. and Schram, A.W. (1987) Eur. J. Biochem. 164, 171-179. 19 Aerts, J.M.F.G., Brul, S., Donker-Koopman, W.E., Van Weely, S., Murray, G.J., Barranger, J.A., Tager, J.M. and Schram, A.W. (1986) Biochem. Biophys. Res. Commun. 141,452-456. 20 Glew, R.H., Daniels, L.B., Clark, L.S. and Hoyer, S.W. (1982) J. Neuropathol. Exp. Neurol. 41,630-641. 21 Bergman, J.E. and Grabowski, G.A. (1989) Am. J. Hum. Genet. 44, 741-750.

22 Van Weely, S., Aerts, J.M.F.G., Van Leeuwen, M.B., Heikoop, J.C., Donker-Koopman, W.E., Barranger, J.A., Tager, J.M. and Schram, A.W. (1990) Eur. J. Biochem. 191,669-677. 23 Willemsen, R., Van Dongen, J.M., Sips, H.J., Schram, A.W., Tager, J.M., Barranger, J.A. and Reuser, A.J.J. (1987) J. Neurol. 234, 44-51. 24 Legler, G. (1977) Methods Enzymol. 46, 368-381. 25 Grabowski, G.A., Goldblatt, J., Dinur, T., Kruse, J., Svennerholm, L., Gatt, S. and Desnick, R.J. (1985) Am. J. Med. Genet. 21, 529-549. 26 Grabowski, G.A., Dinur, T., Osiecki, K.M., Kruse, J.R., Legler, G. and Gatt, S. (1985) Am. J. Hum. Genet. 37, 499-510. 27 Ohashi, T., Hong, C.M., Weiler, S., Tomich, J., Aerts, J., Tager, J. and Barranger, J. (1991) J. Biol. Chem., 264, 3661-3667. 28 Masuno, M., Tomatsu, S., Sukegawa, K. and Orii, T. (1990) Hum. Genet. 84, 203-206. 29 Dahl, N., LagerstriSm, M., Erikson, A. and Pettersson, U. (1990) Am. J. Hum. Genet. 47, 275-278. 30 Sa Miranda, M.C., Aerts, J.M.F.G., Pinto, R., Fontes, A., Wanzeller de Lacerda, L., Van Weely, S., Barranger, J.A. and Tager, J.M. (1990) Clin. Genet. 38, 218-227.